Plant-endophyte combinations and uses therefor

ABSTRACT

The disclosure provides materials and methods for conferring improved plant traits or benefits on plants. The materials can include a formulation comprising an exogenous endophytic bacterial population, which can be disposed on an exterior surface of a seed or seedling, typically in an amount effective to colonize the plant. The formulations can include at least one member selected from the group consisting of an agriculturally compatible carrier, a tackifier, a microbial stabilizer, a fungicide, an antibacterial agent, an herbicide, a nematicide, an insecticide, a plant growth regulator, a rodenticide, and a nutrient.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/166,084, filed May 26, 2016, (allowed), which is a continuation ofU.S. application Ser. No. 14/315,804, filed Jun. 26, 2014, now U.S. Pat.No. 9,364,005, which are incorporated herein by reference for allpurposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing with 10 sequenceswhich has been submitted electronically in ASCII format and is herebyincorporated by reference in its entirety. Said ASCII copy, created onApr. 17, 2019, is named 33748_US_sequencelisting.txt and is 20,480 bytesin size.

BACKGROUND

With limited arable land coupled with rising demand of a steadilyincreasing human population that could reach 9 billion by 2050, foodsupply is a global challenge making production of economicallyattractive and high quality food, free from unacceptable levels ofagrochemicals, a dire need.

Traditional plant breeding strategies to enhance plant traits arerelatively slow and inefficient. For example, breeding plants forincreased tolerance to abiotic stress requires abiotic stress-tolerantfounder lines for crossing with other germplasm to develop new abioticstress-resistant lines. Limited germplasm resources for such founderlines and incompatibility in crosses between distantly related plantspecies represent significant problems encountered in conventionalbreeding. Breeding for stress tolerance has often been inadequate giventhe incidence of stresses and the impact that stresses have on cropyields in most environments of the world.

SUMMARY OF THE INVENTION

The present invention is based on the systematic efforts to discoverendophytic bacterial species that have the potential to greatly improveagricultural productivity. The endophytic bacterial strains extensivelycharacterized herein are able to confer onto the host plant several keyfitness benefits and, as such, offer the possibility of improving yieldsof agricultural crops without the need for time-consuming breedingefforts or genetic modification.

In a first aspect, the present invention provides for an agriculturalplant or portion thereof comprising an exogenous endophytic bacterialpopulation disposed on an exterior surface of the seed or seedling in anamount effective to colonize the plant, and further comprising aformulation that comprises at least one member selected from the groupconsisting of an agriculturally compatible carrier, a tackifier, amicrobial stabilizer, a fungicide, an antibacterial agent, an herbicide,a nematicide, an insecticide, a plant growth regulator, a rodenticide,and a nutrient. The agricultural plant can be a mature plant. In othercases, it can be a seedling. In still other cases, it can be a seed ofan agricultural plant. In one particular embodiment, the agriculturalplant is a seed or seedling.

In one embodiment, the endophytic bacterial population consistsessentially of an endophytic bacterium comprising a 16S rRNA nucleicacid sequence at least 97% identical to a nucleic acid sequence selectedfrom the group consisting of SEQ ID NOs: 1-10.

In one particular embodiment, the endophytic bacterium is a species ofAgrobacterium. In a particular embodiment, the Agrobacterium species isidentified on the basis of its rDNA sequence, as outlined herein. In aparticular embodiment, the Agrobacterium species comprises a 16S rDNAsequence that is at least 97% identical to SEQ ID NO: 1. In anotherembodiment, the Agrobacterium species comprises a 16S rDNA sequence thatis at least 99% identical to SEQ ID NO: 1. In still another embodiment,the Agrobacterium species comprises a 16S rDNA sequence that isidentical to SEQ ID NO: 1. In still another embodiment, theAgrobacterium species is the isolate FA13.

In another embodiment, the endophytic bacterium is a species of Pantoea.In a particular embodiment, the Pantoea species is identified on thebasis of its rDNA sequence, as outlined herein. In a particularembodiment, the Pantoea species comprises a 16S rDNA sequence that is atleast 97%) identical to SEQ ID 2. In another embodiment, the Pantoeaspecies comprises a 16S rDNA sequence that is at least 99% identical toSEQ ID NO: 2. In still another embodiment, the Pantoea species comprisesa 16S rDNA sequence that is identical to SEQ ID NO: 2. In still anotherembodiment, the Pantoea species is the isolate FF34.

In another embodiment, the endophytic bacterium is a species ofSphingobium. In a particular embodiment, the Sphingobium species isidentified on the basis of its rDNA sequence, as outlined herein. In aparticular embodiment, the Sphingobium species comprises a 16S rDNAsequence that is at least 97% identical to SEQ ID NO: 3. In anotherembodiment, the Sphingobium species comprises a 16S rDNA sequence thatis at least 99% identical to SEQ ID NO: 3. In still another embodiment,the Sphingobium species comprises a 16S rDNA sequence that is identicalto SEQ ID NO: 3. In still another embodiment, the Sphingobium species isthe isolate FC42.

In another embodiment, the endophytic bacterium is a species ofPseudomonas. In a particular embodiment, the Pseudomonas species isidentified on the basis of its rDNA sequence, as outlined herein. In aparticular embodiment, the Pseudomonas species comprises a 16S rDNAsequence that is at least 97% identical to SEQ ID NO: 4. In anotherembodiment, the Pseudomonas species comprises a 16S rDNA sequence thatis at least 99% identical to SEQ ID NO: 4. In still another embodiment,the Pseudomonas species comprises a 16S rDNA sequence that is identicalto SEQ ID NO: 4. In still another embodiment, the Pseudomonas species isthe isolate FB12.

In another embodiment, the endophytic bacterium is a species ofEnterobacter. In a particular embodiment, the Enterobacter species isidentified on the basis of its rDNA sequence, as outlined herein. In aparticular embodiment, the Enterobacter species comprises a 16S rDNAsequence that is at least 97% identical to SEQ ID NO: 5. In anotherembodiment, the Enterobacter species comprises a 16S rDNA sequence thatis at least 99% identical to SEQ ID NO: 5. In still another embodiment,the Enterobacter species comprises a 16S rDNA sequence that is identicalto SEQ ID NO: 5. In still another embodiment, the Enterobacter speciesis the isolate FD17.

In another embodiment, the endophytic bacterium is a species ofMicrococcus. In a particular embodiment, the Micrococcus species isidentified on the basis of its rDNA sequence, as outlined herein. In aparticular embodiment, the Micrococcus species comprises a 16S rDNAsequence that is at least 97% identical to SEQ ID NO: 6. In anotherembodiment, the Micrococcus species comprises a 16S rDNA sequence thatis at least 99% identical to SEQ ID NO: 6. In still another embodiment,the Micrococcus species comprises a 16S rDNA sequence that is identicalto SEQ ID NO: 6. In still another embodiment, the Micrococcus species isthe isolate S2.

In another embodiment, the endophytic bacterium is a species ofBacillus. In a particular embodiment, the Bacillus species is identifiedon the basis of its rDNA sequence, as outlined herein. In a particularembodiment, the Bacillus species comprises a 16S rDNA sequence that isat least 97% identical to SEQ ID NO: 7. In another embodiment, theBacillus species comprises a 16S rDNA sequence that is at least 99%identical to SEQ ID NO: 7. In still another embodiment, the Bacillusspecies comprises a 16S rDNA sequence that is identical to SEQ ID NO: 7.In still another embodiment, the Bacillus species is the isolate S4.

In another embodiment, the endophytic bacterium is a species of Pantoea.In a particular embodiment, the Pantoea species is identified on thebasis of its rDNA sequence, as outlined herein. In a particularembodiment, the Pantoea species comprises a 16S rDNA sequence that is atleast 97% identical to SEQ ID NO: 8. In another embodiment, the Pantoeaspecies comprises a 16S rDNA sequence that is at least 99% identical toSEQ ID NO: 8. In still another embodiment, the Pantoea species comprisesa 16S rDNA sequence that is identical to SEQ ID NO: 8. In still anotherembodiment, the Pantoea species is the isolate S6.

In another embodiment, the endophytic bacterium is a species ofAcinetobacter. In a particular embodiment, the Acinetobacter species isidentified on the basis of its rDNA sequence, as outlined herein. In aparticular embodiment, the Acinetobacter species comprises a 16S rDNAsequence that is at least 97% identical to SEQ ID NO: 9. In anotherembodiment, the Acinetobacter species comprises a 16S rDNA sequence thatis at least 99% identical to SEQ ID NO: 9. In still another embodiment,the Acinetobacter species comprises a 16S rDNA sequence that isidentical to SEQ ID NO: 9. In still another embodiment, theAcinetobacter species is the isolate S9.

In another embodiment, the endophytic bacterium is a species ofPaenibacillus. In a particular embodiment, the Paenibacillus species isidentified on the basis of its rDNA sequence, as outlined herein. In aparticular embodiment, the Paenibacillus species comprises a 16S rDNAsequence that is at least 97% identical to SEQ ID NO: 10. In anotherembodiment, the Paenibacillus species comprises a 16S rDNA sequence thatis at least 99% identical to SEQ ID NO: 10. In still another embodiment,the Paenibacillus species comprises a 16S rDNA sequence that isidentical to SEQ ID NO: 10. In still another embodiment, thePaenibacillus species is the isolate S10.

In certain cases, the endophytic bacterial population is disposed in anamount effective to be detectable within a target tissue of the matureagricultural plant selected from a fruit, a seed, a leaf, or a root, orportion thereof.

In certain embodiments, the seed or seedling comprises at least about100 CFU, for example, at least about 200 CFU, at least about 300 CFU, atleast about 500 CFU, at least about 1,000 CFU, at least about 3,000 CFU,at least about 10,000 CFU, at least about 30,000 CFU, at least about100,000 CFU or more, of the endophytic bacterial population on itsexterior surface.

In another embodiment, the endophytic bacterial population is disposedon an exterior surface or within a tissue of the seed or seedling in anamount effective to be detectable in an amount of at least about 100CFU, for example, at least about 200 CFU, at least about 300 CFU, atleast about 500 CFU, at least about 1,000 CFU, at least about 3,000 CFU,at least about 10,000 CFU, at least about 30,000 CFU, at least about100,000 CFU or more per gram fresh weight of the mature agriculturalplant.

In another embodiment, the endophytic bacterial population is disposedon the surface or within a tissue of the seed or seedling in an amounteffective to increase the biomass of the fruit or cob from the resultingplant by at least 10% when compared with a reference agricultural plant.

In still another embodiment, the endophytic bacterial population isdisposed on the surface or within a tissue of the seed or seedling in anamount effective to detectably colonize the soil environment surroundingthe mature agricultural plant when compared with a referenceagricultural plant.

In some cases, the endophytic bacterial population is disposed in anamount effective to increase root biomass by at least 10% when comparedwith a reference agricultural plant.

In some embodiments, the endophytic bacterial population is disposed onthe surface or within a tissue of the seed or seedling in an amounteffective to increase the rate of seed germination when compared with areference agricultural plant.

In another embodiment, the endophytic bacterial population is disposedon the surface or within a tissue of the seed or seedling in an amounteffective to detectably induce production of auxin in the seed orseedling.

In one embodiment, the endophytic bacterial population is cultured priorto disposition on the seed or seedling. In one embodiment, theendophytic bacterial population is cultured in a synthetic orsemi-synthetic medium prior to disposition on the seed or seedling.

In certain cases, the endophytic bacterial population can be modified.In one embodiment, the endophytic bacterial population is geneticallymodified. In another embodiment, the endophytic bacterial population ismodified such that it has enhanced compatibility with an antimicrobialagent when compared with an unmodified control. The antimicrobial agentis an antibacterial agent. Alternatively, the antimicrobial agent can bean antifungal agent. In some cases, the modified endophytic bacterialpopulation exhibits at least 3 fold greater, for example, at least 5fold greater, at least 10 fold greater, at least 20 fold greater, atleast 30 fold greater or more resistance to the antimicrobial agent whencompared with an unmodified control. In one embodiment, theantimicrobial agent is glyphosate.

The seed or seedling of the agricultural plant can be a monocot. Forexample, it can be a corn seed or seedling. Alternatively, it can be awheat seed or seedling. In other embodiments, it can be a barley seed orseedling. In still other cases, it can be a rice seed or seedling.

In another embodiment, the seed or seedling is a dicot. For example, itcan be a cotton seed or seedling, a soy seed or seedling, or a tomatoseed or seedling.

In still another embodiment, the seed or seedling can be derived from atransgenic plant. In another embodiment, the seed or seedling can be ahybrid seed or seedling.

In one particular embodiment, the seed is a corn seed, and furthercomprises at least about 10,000 CFU of the endophytic bacterialpopulation consisting essentially of an endophytic bacterium comprisinga 16S rRNA nucleic acid sequence that is at least 97%, for example, atleast 99%, at least 99.5%), or 100%) identical to a nucleic acidsequence selected from the group consisting of [SEQ ID NOs: 1-10]disposed on the exterior surface of the seed, and further comprising aformulation comprising a microbial stabilizer.

In another aspect, the invention provides for a substantially uniformpopulation of seeds comprising a plurality of seeds described above.Substantial uniformity can be determined in many ways. In some cases, atleast 10%, for example, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 75%, at least 80%, atleast 90%, at least 95% or more of the seeds in the population, containsthe endophytic bacterial population in an amount effective to colonizethe plant disposed on the surface of the seeds. In other cases, at least10%, for example, at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 75%, at least 80%), at least90%, at least 95% or more of the seeds in the population, contains atleast 100 CFU on its surface, for example, at least 200 CFU, at least300 CFU, at least 1,000 CFU, at least 3,000 CFU, at least 10,000 CFU, atleast 30,000 CFU, at least 100,000 CFU, at least 300,000 CFU, or atleast 1,000,000 CFU per seed or more.

In yet another aspect, the present invention provides for a bagcomprising at least 1,000 seeds as described herein above. The bagfurther comprises a label describing the seeds and/or said endophyticbacterial population.

In still another aspect of the present invention, a plant or part ortissue of the plant, or progeny thereof is disclosed, which is generatedby growing the seed or seedling described herein above.

In yet another aspect, disclosed are substantially uniform populationsof plants produced by growing a plurality of seeds, seedlings, orprogeny thereof. In some cases, at least 75%, at least 80%, at least90%, at least 95% or more of the plants in the population comprise anamount of the endophytic bacterial population effective to increase theroot biomass of the plant by at least 10%. In other cases, at least 10%,for example at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%), at least 70%, at least 75%, at least 80%, at least 90%, atleast 95% or more of the plants comprise a microbe population that issubstantially similar.

In yet another aspect of the present invention, disclosed is anagricultural field comprising the population described above. The fieldgenerally comprises at least 100 plants, for example, at least 1,000plants, at least 3,000 plants, at least 10,000 plants, at least 30,000plants, at least 100,000 plants or more in the field. In certain cases,the population of plants occupies at least about 100 square feet ofspace, and at least about 10%, for example, at least 20%, at least 30%,at least 40%, at least 50%), at least 60%, at least 70%, at least 80%,at least 90% or more than 90% of the population comprises an amount ofthe endophytic bacterial population effective to increase the rootbiomass of the plant by at least 10%. In another embodiment, thepopulation of plants occupies at least about 100 square feet of space,wherein and at least about 10%, for example, at least 20%, at least 30%,at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90% or more than 90% of the population comprises the microbe inreproductive tissue. In another embodiment, the population of plantsoccupies at least about 100 square feet of space, and at least about10%, for example, at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90% or more than90% of the population comprises at least 100 CFUs, 1,000 CFUs, 10,000CFUs, 100,000 CFUs or more of the endophytic bacterial population.

In another aspect of the invention, provided are preparations comprisinga population of endophytic bacteria described herein and furthercomprising at least one agent selected from the group consisting of anagriculturally acceptable carrier, a tackifier, a microbial stabilizer,a fungicide, an antibacterial agent, an herbicide, a nematicide, aninsecticide, a plant growth regulator, a rodenticide, and a nutrient,and wherein the population comprises an amount of endophytes sufficientto improve an agronomic trait of the population of seeds. In oneembodiment, the endophytic bacterial population consists essentially ofan endophytic bacterium comprising a 16S rRNA nucleic acid sequence atleast 97% identical to a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs: 1-10.

In one embodiment, the preparation is substantially stable attemperatures between about 4° C. and about 45° C. for at least aboutseven days.

In another embodiment, the preparation is formulated to provide at least100 endophytes per seed, for example, at least 300 endophytes, at least1,000 endophytes, at least 3,000 endophytes, at least 10,000 endophytes,at least 30,000 endophytes, at least 100,000 endophytes, at least300,000 endophytes, or at least 1,000,000 endophytes per seed.

In another embodiment, the preparation is formulated to provide apopulation of plants that demonstrates a substantially homogenous growthrate when introduced into agricultural production.

In still another aspect, the present invention provides for a method ofproducing a commodity plant product. The method generally comprisesobtaining a plant or plant tissue from the agricultural plant comprisingthe endophytic bacteria as described herein above, and producing thecommodity plant product therefrom. In certain cases, the commodity plantproduct is selected from the group consisting of grain, flour, starch,seed oil, syrup, meal, flour, oil, film, packaging, nutraceuticalproduct, an animal feed, a fish fodder, a cereal product, a processedhuman-food product, a sugar or an alcohol and protein.

In a related aspect, the present invention provides for a commodityplant product comprising a plant or part thereof and further comprisingthe endophytic bacterial population or a portion thereof in a detectablelevel.

In yet another aspect of the present invention, provided is a method forpreparing an agricultural plant or a portion thereof comprising anendophytic bacterial population. The method generally comprises applyingto the seed or seedling a formulation comprising an endophytic bacterialpopulation consisting essentially of an endophytic bacterium comprisinga 16S rRNA nucleic acid sequence at least 97% identical, for example, atleast 98% identical, at least 99% identical, at least 99.5% identical,or 100% identical to a nucleic acid sequence selected from the groupconsisting of [SEQ ID NOs: 1-10]. In one embodiment, the formulationfurther comprises at least one member selected from the group consistingof an agriculturally compatible carrier, a tackifier, a microbialstabilizer, a fungicide, an antibacterial agent, an herbicide, anematicide, an insecticide, a plant growth regulator, a rodenticide, anda nutrient. In some cases, the agricultural plant can be a seedling. Inother cases, the agricultural plant can be a seed. In a particularembodiment, the agricultural plant is a seed or a seedling. In anotherembodiment, the method further comprises applying at least one memberselected from the group consisting of an agriculturally compatiblecarrier, a tackifier, a microbial stabilizer, a fungicide, anantibacterial agent, an herbicide, a nematicide, an insecticide, a plantgrowth regulator, a rodenticide, and a nutrient.

In a final aspect, the present invention provides for a method forconferring one or more fitness benefits to an agricultural plant. Themethod generally comprises providing an agricultural plant or portionthereof, contacting said plant or portion thereof with a formulationcomprising an exogenous endophytic bacterial population consistingessentially of an endophytic bacterium comprising a 16S rRNA nucleicacid sequence at least 97% identical, for example, at least 98%identical, at least 99% identical, at least 99.5% identical, or 100%identical to a nucleic acid sequence selected from the group consistingof [SEQ ID NOs: 1-10], disposed on an exterior surface in an amounteffective to colonize the mature plant, wherein the formulation furthercomprises at least one member selected from the group consisting of anagriculturally compatible carrier, a tackifier, a microbial stabilizer,a fungicide, an antibacterial agent, an herbicide, a nematicide, aninsecticide, a plant growth regulator, a rodenticide, and a nutrient,and allowing the seed or seedling to grow under conditions that allowthe endophytic bacterium to colonize the plant. In some cases, theagricultural plant can be a seedling. In other cases, the agriculturalplant can be a seed. In a particular embodiment, the agricultural plantis a seed or a seedling.

In one embodiment, the one or more of the fitness benefits are selectedfrom the group consisting of increased germination, increased biomass,increased flowering time, increased biomass of the fruit or grain,increased grain or fruit yield, and increased drought tolerance.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A, FIG. 1B, and FIG. 1C each show the increases in root and shootlength in maize plants inoculated with the bacterial endophytepopulations when compared with uninoculated control plants.

FIG. 2 shows the increases in stomatal conductance in maize plantsinoculated with the bacterial endophyte populations when compared withuninoculated control plants.

FIG. 3 shows the increase in photosynthetic rates in maize plantsinoculated with the bacterial endophyte populations when compared withuninoculated control plants.

FIG. 4 shows the increases in PS II photochemical efficiency (Fv/Fm) inmaize plants inoculated with the bacterial endophyte populations, whencompared with uninoculated control plants.

FIG. 5 shows the increases in leaf area in maize plants inoculated withthe bacterial endophyte populations, when compared with uninoculatedcontrol plants.

FIG. 6 shows the increases in chlorophyll content in maize plantsinoculated with the bacterial endophyte populations, when compared withuninoculated control plants.

DETAILED DESCRIPTION

Definitions

A “synthetic combination” includes a combination of a host plant and anendophyte. The combination may be achieved, for example, by coating thesurface of the seed of a plant, such as an agricultural plant, or hostplant tissues with an endophyte.

As used herein, an “agricultural seed” is a seed used to grow a plant inagriculture (an “agricultural plant”). The seed may be of a monocot ordicot plant, and is planted for the production of an agriculturalproduct, for example grain, food, fiber, etc. As used herein, anagricultural seed is a seed that is prepared for planting, for example,in farms for growing.

An “endophyte”, or “endophytic microbe” includes an organism capable ofliving within a plant or associated therewith. An endophyte may refer toa bacterial or fungal organism that may confer an increase in yield,biomass, resistance, or fitness in its host plant. Endophytes may occupythe intracellular or extracellular spaces of plant tissue, including theleaves, stems, flowers, fruits, seeds, or roots. An endophyte can be afungus, or a bacterium. As used herein, the term “microbe” is sometimesused to describe an endophyte.

In some embodiments, the invention contemplates the use of microbes thatare “exogenous” to a seed or plant. As used herein, a microbe isconsidered exogenous to the seed or plant if the seed or seedling thatis unmodified (e.g., a seed or seedling that is not treated with theendophytic bacterial population descried herein) does not contain themicrobe.

In other cases, the invention contemplates the synthetic combinations ofagricultural plants and an endophytic microbe population, in which themicrobe population is “heterologously disposed” on the surface of orwithin a tissue of the agricultural plant. As used herein, a microbe isconsidered “heterologously disposed” on the surface or within a plant(or tissue) when the microbe is applied or disposed on the plant in anumber or within a tissue in a number that is not found on that plantprior to application of the microbe. As such, a microbe is deemedheterologously disposed when applied on the plant that either does notnaturally have the microbe on its surface or within the particulartissue to which the microbe is disposed, or does not naturally have themicrobe on its surface or within the particular tissue in the numberthat is being applied. For the avoidance of doubt, “heterologouslydisposed” contemplates use of microbes that are “exogenous” to a seed orplant.

In some cases, the present invention contemplates the use of microbesthat are “compatible” with agricultural chemicals for example, afungicide, an anti-bacterial compound, or any other agent widely used inagricultural which has the effect of interfering with optimal growth ofmicrobes. As used herein, a microbe is “compatible” with an agriculturalchemical, when the microbe is modified or otherwise adapted to grow in,or otherwise survive, the concentration of the agricultural chemicalused in agriculture. For example, a microbe disposed on the surface of aseed is compatible with the fungicide metalaxyl if it is able to survivethe concentrations that are applied on the seed surface.

“Biomass” means the total mass or weight (fresh or dry), at a giventime, of a plant tissue, plant tissues, an entire plant, or populationof plants, usually given as weight per unit area. The term may alsorefer to all the plants or species in the community (community biomass).

Some of the compositions and methods described herein involve endophyticmicrobes in an amount effective to colonize a plant. As used herein, amicrobe is said to “colonize” a plant or seed when it can exist in anendophytic relationship with the plant in the plant environment, forexample inside the plant or a part or tissue thereof, including theseed.

Some compositions described herein contemplate the use of anagriculturally compatible carrier. As used herein an “agriculturallycompatible carrier” is intended to refer to any material, other thanwater, which can be added to a seed or a seedling without causing/havingan adverse effect on the seed, the plant that grows from the seed, seedgermination, or the like.

A “transgenic plant” includes a plant or progeny plant of any subsequentgeneration derived therefrom, wherein the DNA of the plant or progenythereof contains an introduced exogenous DNA segment not naturallypresent in a non-transgenic plant of the same strain. The transgenicplant may additionally contain sequences that are native to the plantbeing transformed, but wherein the “exogenous” gene has been altered inorder to alter the level or pattern of expression of the gene, forexample, by use of one or more heterologous regulatory or otherelements.

As used herein, a nucleic acid has “homology” or is “homologous” to asecond nucleic acid if the nucleic acid sequence has a similar sequenceto the second nucleic acid sequence. The terms “identity”, “percentsequence identity” or “identical” in the context of nucleic acidsequences refer to the residues in the two sequences that are the samewhen aligned for maximum correspondence. There are a number of differentalgorithms known in the art that can be used to measure nucleotidesequence identity. For instance, polynucleotide sequences can becompared using FASTA, Gap or Bestfit, which are programs in WisconsinPackage Version 10.0, Genetics Computer Group (GCG), Madison, Wis. FASTAprovides alignments and percent sequence identity of the regions of thebest overlap between the query and search sequences. Pearson, MethodsEnzymol. 183:63-98 (1990). The term “substantial homology” or“substantial similarity,” when referring to a nucleic acid or fragmentthereof, indicates that, when optimally aligned with appropriatenucleotide insertions or deletions with another nucleic acid (or itscomplementary strand), there is nucleotide sequence identity in at leastabout 76%, 80%, 85%, or at least about 90%, or at least about 95%, 96%,97%, 98%) or 99% of the nucleotide bases, as measured by any well-knownalgorithm of sequence identity, such as FASTA, BLAST or Gap, asdiscussed above.

The present invention is directed to methods and compositions ofbacterial endophytes, and plant-endophyte combinations that confer afitness benefit in agricultural plants.

Bacterial Endophyte

In a first aspect, disclosed is a composition comprising a pure cultureof a bacterial endophyte.

In one embodiment, the endophytic bacterium is a species ofAgrobacterium. In a particular embodiment, the Agrobacterium species isidentified on the basis of its rDNA sequence, as outlined herein. In aparticular embodiment, the Agrobacterium species comprises a 16S rDNAsequence that is at least 97% identical to SEQ ID NO: 1. In anotherembodiment, the Agrobacterium species comprises a 16S rDNA sequence thatis at least 99% identical to SEQ ID NO: 1. In still another embodiment,the Agrobacterium species comprises a 16S rDNA sequence that isidentical to SEQ ID NO: 1. In still another embodiment, theAgrobacterium species is the isolate FA13.

In another embodiment, the endophytic bacterium is a species of Pantoea.In a particular embodiment, the Pantoea species is identified on thebasis of its rDNA sequence, as outlined herein. In a particularembodiment, the Pantoea species comprises a 16S rDNA sequence that is atleast 97% identical to SEQ ID NO: 2. In another embodiment, the Pantoeaspecies comprises a 16S rDNA sequence that is at least 99% identical toSEQ ID NO: 2. In still another embodiment, the Pantoea species comprisesa 16S rDNA sequence that is identical to SEQ ID NO: 2. In still anotherembodiment, the Pantoea species is the isolate FF34.

In another embodiment, the endophytic bacterium is a species ofSphingobium. In a particular embodiment, the Sphingobium species isidentified on the basis of its rDNA sequence, as outlined herein. In aparticular embodiment, the Sphingobium species comprises a 16S rDNAsequence that is at least 97% identical to SEQ ID NO: 3. In anotherembodiment, the Sphingobium species comprises a 16S rDNA sequence thatis at least 99% identical to SEQ ID NO: 3. In still another embodiment,the Sphingobium species comprises a 16S rDNA sequence that is identicalto SEQ ID NO: 3. In still another embodiment, the Sphingobium species isthe isolate FC42.

In another embodiment, the endophytic bacterium is a species ofPseudomonas. In a particular embodiment, the Pseudomonas species isidentified on the basis of its rDNA sequence, as outlined herein. In aparticular embodiment, the Pseudomonas species comprises a 16S rDNAsequence that is at least 97% identical to SEQ ID NO: 4. In anotherembodiment, the Pseudomonas species comprises a 16S rDNA sequence thatis at least 99% identical to SEQ ID NO: 4. In still another embodiment,the Pseudomonas species comprises a 16S rDNA sequence that is identicalto SEQ ID NO: 4. In still another embodiment, the Pseudomonas species isthe isolate FB12.

In another embodiment, the endophytic bacterium is a species ofEnterobacter. In a particular embodiment, the Enterobacter species isidentified on the basis of its rDNA sequence, as outlined herein. In aparticular embodiment, the Enterobacter species comprises a 16S rDNAsequence that is at least 97% identical to SEQ ID NO: 5. In anotherembodiment, the Enterobacter species comprises a 16S rDNA sequence thatis at least 99% identical to SEQ ID NO: 5. In still another embodiment,the Enterobacter species comprises a 16S rDNA sequence that is identicalto SEQ ID NO: 5. In still another embodiment, the Enterobacter speciesis the isolate FD17.

In another embodiment, the endophytic bacterium is a species ofMicrococcus. In a particular embodiment, the Micrococcus species isidentified on the basis of its rDNA sequence, as outlined herein. In aparticular embodiment, the Micrococcus species comprises a 16S rDNAsequence that is at least 97% identical to SEQ ID NO: 6. In anotherembodiment, the Micrococcus species comprises a 16S rDNA sequence thatis at least 99% identical to SEQ ID NO: 6. In still another embodiment,the Micrococcus species comprises a 16S rDNA sequence that is identicalto SEQ ID NO: 6. In still another embodiment, the Micrococcus species isthe isolate S2.

In another embodiment, the endophytic bacterium is a species ofBacillus. In a particular embodiment, the Bacillus species is identifiedon the basis of its rDNA sequence, as outlined herein. In a particularembodiment, the Bacillus species comprises a 16S rDNA sequence that isat least 97% identical to SEQ ID NO: 7. In another embodiment, theBacillus species comprises a 16S rDNA sequence that is at least 99%identical to SEQ ID NO: 7. In still another embodiment, the Bacillusspecies comprises a 16S rDNA sequence that is identical to SEQ ID NO: 7.In still another embodiment, the Bacillus species is the isolate S4.

In another embodiment, the endophytic bacterium is a species of Pantoea.In a particular embodiment, the Pantoea species is identified on thebasis of its rDNA sequence, as outlined herein. In a particularembodiment, the Pantoea species comprises a 16S rDNA sequence that is atleast 97%) identical to SEQ ID NO: 8. In another embodiment, the Pantoeaspecies comprises a 16S rDNA sequence that is at least 99% identical toSEQ ID NO: 8. In still another embodiment, the Pantoea species comprisesa 16S rDNA sequence that is identical to SEQ ID NO: 8. In still anotherembodiment, the Pantoea species is the isolate S6.

In another embodiment, the endophytic bacterium is a species ofAcinetobacter. In a particular embodiment, the Acinetobacter species isidentified on the basis of its rDNA sequence, as outlined herein. In aparticular embodiment, the Acinetobacter species comprises a 16S rDNAsequence that is at least 97% identical to SEQ ID NO: 9. In anotherembodiment, the Acinetobacter species comprises a 16S rDNA sequence thatis at least 99% identical to SEQ ID NO: 9. In still another embodiment,the Acinetobacter species comprises a 16S rDNA sequence that isidentical to SEQ ID NO: 9. In still another embodiment, theAcinetobacter species is the isolate S9.

In another embodiment, the endophytic bacterium is a species ofPaenibacillus. In a particular embodiment, the Paenibacillus species isidentified on the basis of its rDNA sequence, as outlined herein. In aparticular embodiment, the Paenibacillus species comprises a 16S rDNAsequence that is at least 97% identical to SEQ ID NO: 10. In anotherembodiment, the Paenibacillus species comprises a 16S rDNA sequence thatis at least 99% identical to SEQ ID NO: 10. In still another embodiment,the Paenibacillus species comprises a 16S rDNA sequence that isidentical to SEQ ID NO: 10. In still another embodiment, thePaenibacillus species is the isolate S10.

In some cases, the endophytic microbe can be modified. For example, theendophytic microbe can be genetically modified by introduction of atransgene which stably integrates into the bacterial genome. In anotherembodiment, the endophytic microbe can be modified to harbor a plasmidor episome containing a transgene. In still another embodiment, themicrobe can be modified by repeated passaging under selectiveconditions.

The microbe can be modified to exhibit altered characteristics. In oneembodiment, the endophytic microbe is modified to exhibit increasedcompatibility with chemicals commonly used in agriculture. Agriculturalplants are often treated with a vast array of agrichemicals, includingfungicides, biocides (anti-bacterial agents), herbicides, insecticides,nematicides, rodenticides, fertilizers, and other agents. Many suchagents can affect the ability of an endophytic bacterium to grow,divide, and/or otherwise confer beneficial traits to the plant.

In some cases, it can be important for the microbe to be compatible withagrichemicals, particularly those with fungicidal or antibacterialproperties, in order to persist in the plant although, as mentionedearlier, there are many such fungicidal or antibacterial agents that donot penetrate the plant, at least at a concentration sufficient tointerfere with the microbe. Therefore, where a systemic fungicide orantibacterial agent is used in the plant, compatibility of the microbeto be inoculated with such agents will be an important criterion.

In one embodiment, spontaneous isolates of microbes which are compatiblewith agrichemicals can be used to inoculate the plants according to themethods described herein. For example, fungal microbes which arecompatible with agriculturally employed fungicides can be isolated byplating a culture of the microbes on a petri dish containing aneffective concentration of the fungicide, and isolating colonies of themicrobe that are compatible with the fungicide. In another embodiment, amicrobe that is compatible with a fungicide is used for the methodsdescribed herein. In still another embodiment, a microbe that iscompatible with an antibacterial compound is used for the methodsdescribed herein. Fungicide compatible microbes can also be isolated byselection on liquid medium. The culture of microbes can be plated onpetri dishes without any forms of mutagenesis; alternatively, themicrobes can be mutagenized using any means known in the art. Forexample, microbial cultures can be exposed to UV light,gamma-irradiation, or chemical mutagens such as ethylmethanesulfonate(EMS) prior to selection on fungicide containing media.

Finally, where the mechanism of action of a particular fungicide isknown, the target gene can be specifically mutated (either by genedeletion, gene replacement, site-directed mutagenesis, etc.) to generatea microbe that is resilient against that particular fungicide. It isnoted that the above-described methods can be used to isolate fungi thatare compatible with both fungistatic and fungicidal compounds.

It will also be appreciated by one skilled in the art that a plant maybe exposed to multiple types of fungicides or antibacterial compounds,either simultaneously or in succession, for example at different stagesof plant growth. Where the target plant is likely to be exposed tomultiple fungicidal and/or antibacterial agents, a microbe that iscompatible with many or all of these agrichemicals can be used toinoculate the plant. A microbe that is compatible with severalfungicidal agents can be isolated, for example, by serial selection. Amicrobe that is compatible with the first fungicidal agent is isolatedas described above (with or without prior mutagenesis). A culture of theresulting microbe can then be selected for the ability to grow on liquidor solid media containing the second antifungal compound (again, with orwithout prior mutagenesis). Colonies isolated from the second selectionare then tested to confirm its compatibility to both antifungalcompounds.

Likewise, bacterial microbes that are compatible to biocides (includingherbicides such as glyphosate or antibacterial compounds, whetherbacteriostatic or bactericidal) that are agriculturally employed can beisolated using methods similar to those described for isolatingfungicide compatible microbes. In one embodiment, mutagenesis of themicrobial population can be performed prior to selection with anantibacterial agent. In another embodiment, selection is performed onthe microbial population without prior mutagenesis. In still anotherembodiment, serial selection is performed on a microbe: the microbe isfirst selected for compatibility to a first antibacterial agent. Theisolated compatible microbe is then cultured and selected forcompatibility to the second antibacterial agent. Any colony thusisolated is tested for compatibility to each, or both antibacterialagents to confirm compatibility with these two agents.

The selection process described above can be repeated to identifyisolates of the microbe that are compatible with a multitude ofantifungal or antibacterial agents.

Candidate isolates can be tested to ensure that the selection foragrichemical compatibility did not result in loss of a desired microbialbioactivity. Isolates of the microbe that are compatible with commonlyemployed fungicides can be selected as described above. The resultingcompatible microbe can be compared with the parental microbe on plantsin its ability to promote germination.

Plant-Endophyte Combinations

In another aspect, the present invention provides for combinations ofendophytes and plants. In one embodiment, disclosed is a seed orseedling of an agricultural plant comprising an exogenous endophyticbacterial population that is disposed on an exterior surface of orwithin the seed or seedling in an amount effective to colonize theplant, and further comprising a formulation that comprises at least onemember selected from the group consisting of an agriculturallycompatible carrier, a tackifier, a microbial stabilizer, a fungicide, anantibacterial agent, an herbicide, a nematicide, an insecticide, a plantgrowth regulator, a rodenticide, and a nutrient. In another embodiment,the present invention discloses a seed or seedling of an agriculturalplant comprising an endophytic bacterial population that isheterologously disposed on an exterior surface of or within the seed orseedling in an amount effective to colonize the plant, and furthercomprising a formulation that comprises at least one member selectedfrom the group consisting of an agriculturally compatible carrier, atackifier, a microbial stabilizer, a fungicide, an antibacterial agent,an herbicide, a nematicide, an insecticide, a plant growth regulator, arodenticide, and a nutrient.

The endophytic bacterial population consists essentially of anendophytic bacterium described herein. In one embodiment, the endophyticbacterium comprises a 16S rRNA nucleic acid sequence that is at least97% identical to a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs: 1-10. In another embodiment, the endophyticbacterium comprises a 16S rRNA nucleic acid sequence that is at least99% identical to a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs: 1-10. In still another embodiment, theendophytic bacterium comprises a 16S rRNA nucleic acid sequence that isidentical to a nucleic acid sequence selected from the group consistingof SEQ ID NOs: 1-10.

In one embodiment according to this aspect, disclosed is a seed of anagricultural plant comprising an exogenous endophytic bacterialpopulation that is disposed on an exterior surface of or within the seedin an amount effective to colonize the plant. The bacterial populationis considered exogenous to the seed if that particular seed does notinherently contain the bacterial population. Indeed, several of theendophytic microbes described herein have not been detected, forexample, in any of the corn seeds sampled, as determined by highlysensitive methods.

In other cases, the present invention discloses a seed of anagricultural plant comprising an endophytic bacterial population that isheterologously disposed on an exterior surface of or within the seed inan amount effective to colonize the plant. For example, the endophyticbacterial population that is disposed on an exterior surface or withinthe seed can be an endophytic bacterium that may be associated with themature plant, but is not found on the surface of or within the seed.Alternatively, the endophytic bacterial population can be found in thesurface of, or within the seed, but at a much lower number than isdisposed.

As shown in the Examples section below, the endophytic bacterialpopulations described herein are capable of colonizing the host plant.In certain cases, the endophytic bacterial population can be applied tothe plant, for example the plant seed, or by foliar application, andsuccessful colonization can be confirmed by detecting the presence ofthe bacterial population within the plant. For example, after applyingthe bacteria to the seeds, high titers of the bacteria can be detectedin the roots and shoots of the plants that germinate from the seeds. Inaddition, significant quantities of the bacteria can be detected in therhizosphere of the plants. Therefore, in one embodiment, the endophyticmicrobe population is disposed in an amount effective to colonize theplant. Colonization of the plant can be detected, for example, bydetecting the presence of the endophytic microbe inside the plant. Thiscan be accomplished by measuring the viability of the microbe aftersurface sterilization of the seed or the plant: endophytic colonizationresults in an internal localization of the microbe, rendering itresistant to conditions of surface sterilization. The presence andquantity of the microbe can also be established using other means knownin the art, for example, immunofluorescence microscopy using microbespecific antibodies, or fluorescence in situ hybridization (see, forexample, Amann et al. (2001) Current Opinion in Biotechnology12:231-236, incorporated herein by reference in its entirety).Alternatively, specific nucleic acid probes recognizing conservedsequences from the endophytic bacterium can be employed to amplify aregion, for example by quantitative PCR, and correlated to CFUs by meansof a standard curve.

In another embodiment, the endophytic microbe is disposed in an amounteffective to be detectable in the mature agricultural plant. In oneembodiment, the endophytic microbe is disposed in an amount effective tobe detectable in an amount of at least about 100 CFU, at least about 200CFU, at least about 300 CFU, at least about 500 CFU, at least about1,000 CFU, at least about 3,000 CFU, at least about 10,000 CFU, at leastabout 30,000 CFU, at least about 100,000 CFU or more in the matureagricultural plant.

In some cases, the endophytic microbe is capable of colonizingparticular tissue types of the plant. In one embodiment, the endophyticmicrobe is disposed on the seed or seedling in an amount effective to bedetectable within a target tissue of the mature agricultural plantselected from a fruit, a seed, a leaf, or a root, or portion thereof.For example, the endophytic microbe can be detected in an amount of atleast about 100 CFU, at least about 200 CFU, at least about 300 CFU, atleast about 500 CFU, at least about 1,000 CFU, at least about 3,000 CFU,at least about 10,000 CFU, at least about 30,000 CFU, at least about100,000 CFU or more, in the target tissue of the mature agriculturalplant.

In some cases, the microbes disposed on the seed or seedling can bedetected in the rhizosphere. This may be due to successful colonizationby the endophytic microbe, where certain quantities of the microbe isshed from the root, thereby colonizing the rhizosphere. In some cases,the rhizosphere-localized microbe can secrete compounds (such assiderophores or organic acids) which assist with nutrient acquisition bythe plant. Therefore, in another embodiment, the endophytic microbe isdisposed on the surface of the seed in an amount effective to detectablycolonize the soil environment surrounding the mature agricultural plantwhen compared with a reference agricultural plant. For example, themicrobe can be detected in an amount of at least 100 CFU/g DW, forexample, at least 200 CFU/g DW, at least 500 CFU/g DW, at least 1,000CFU/g DW, at least 3,000 CFU/g DW, at least 10,000 CFU/g DW, at least30,000 CFU/g DW, at least 100,000 CFU/g DW, at least 300,000 CFU/g DW,or more, in the rhizosphere.

The endophytic bacterial populations described herein are also capableof providing many fitness benefits to the host plant. As shown in theExamples section, endophyte-inoculated plants display increased seedgermination, increased vigor, increased biomass (e.g., increased root orshoot biomass), increased photochemical efficiency. Therefore, in oneembodiment, the endophytic bacterial population is disposed on thesurface or within a tissue of the seed or seedling in an amounteffective to increase the biomass of the plant, or a part or tissue ofthe plant grown from the seed or seedling. The increased biomass isuseful in the production of commodity products derived from the plant.Such commodity products include an animal feed, a fish fodder, a cerealproduct, a processed human-food product, a sugar or an alcohol. Suchproducts may be a fermentation product or a fermentable product, onesuch exemplary product is a biofuel. The increase in biomass can occurin a part of the plant (e.g., the root tissue, shoots, leaves, etc.), orcan be an increase in overall biomass. Increased biomass production,such an increase meaning at least about 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 100%, or greater than 100% when compared with a referenceagricultural plant. Such increase in overall biomass can be underrelatively stress-free conditions. In other cases, the increase inbiomass can be in plants grown under any number of abiotic or bioticstresses, including drought stress, salt stress, heat stress, coldstress, low nutrient stress, nematode stress, insect herbivory stress,fungal pathogen stress, bacterial pathogen stress, and viral pathogenstress. In one particular embodiment, the endophytic bacterialpopulation is disposed in an amount effective to increase root biomassby at least 10%, for example, at least 20%, at least 30%, at least 40%,at least 50%), at least 60%, at least 75%, at least 100%, or more, whencompared with a reference agricultural plant.

In another embodiment, the endophytic bacterial population is disposedon the surface or within a tissue of the seed or seedling in an amounteffective to increase the rate of seed germination when compared with areference agricultural plant. For example, the increase in seedgermination can be at least 10%, for example, at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 75%, at least100%, or more, when compared with a reference agricultural plant.

In other cases, the endophytic microbe is disposed on the seed orseedling in an amount effective to increase the average biomass of thefruit or cob from the resulting plant by at least 10%, for example, atleast 20%, at least 30%, at least 40%, at least 50%, at least 75%, atleast 100% or more, when compared with a reference agricultural plant.

As highlighted in the Examples section, plants inoculated with theendophytic bacterial population also show an increase in overall plantheight. Therefore, in one embodiment, the present invention provides fora seed comprising an endophytic bacterial population which is disposedon the surface or within a tissue of the seed or seedling in an amounteffective to increase the height of the plant. For example, theendophytic bacterial population is disposed in an amount effective toresult in an increase in height of the agricultural plant such that isat least 10% greater, for example, at least 20% greater, at least 30%greater, at least 40% greater, at least 50% greater, at least 60%greater, at least 70% greater, at least 80% greater, at least 90%greater, at least 100% greater, at least 125%) greater, at least 150%greater or more, when compared with a reference agricultural plant, theplant. Such increase in height can be under relatively stress-freeconditions. In other cases, the increase in height can be in plantsgrown under any number of abiotic or biotic stresses, including droughtstress, salt stress, heat stress, cold stress, low nutrient stress,nematode stress, insect herbivory stress, fungal pathogen stress,bacterial pathogen stress, and viral pathogen stress.

The host plants inoculated with the endophytic bacterial population alsoshow dramatic improvements in their ability to utilize water moreefficiently. Water use efficiency is a parameter often correlated withdrought tolerance. Water use efficiency (WUE) is a parameter oftencorrelated with drought tolerance, and is the CO2 assimilation rate perwater transpired by the plant. An increase in biomass at low wateravailability may be due to relatively improved efficiency of growth orreduced water consumption. In selecting traits for improving crops, adecrease in water use, without a change in growth would have particularmerit in an irrigated agricultural system where the water input costswere high. An increase in growth without a corresponding jump in wateruse would have applicability to all agricultural systems. In manyagricultural systems where water supply is not limiting, an increase ingrowth, even if it came at the expense of an increase in water use alsoincreases yield.

When soil water is depleted or if water is not available during periodsof drought, crop yields are restricted. Plant water deficit develops iftranspiration from leaves exceeds the supply of water from the roots.The available water supply is related to the amount of water held in thesoil and the ability of the plant to reach that water with its rootsystem. Transpiration of water from leaves is linked to the fixation ofcarbon dioxide by photosynthesis through the stomata. The two processesare positively correlated so that high carbon dioxide influx throughphotosynthesis is closely linked to water loss by transpiration. Aswater transpires from the leaf, leaf water potential is reduced and thestomata tend to close in a hydraulic process limiting the amount ofphotosynthesis. Since crop yield is dependent on the fixation of carbondioxide in photosynthesis, water uptake and transpiration arecontributing factors to crop yield. Plants which are able to use lesswater to fix the same amount of carbon dioxide or which are able tofunction normally at a lower water potential have the potential toconduct more photosynthesis and thereby to produce more biomass andeconomic yield in many agricultural systems. An increased water useefficiency of the plant relates in some cases to an increasedfruit/kernel size or number.

Therefore, in one embodiment, the plants described herein exhibit anincreased water use efficiency when compared with a referenceagricultural plant grown under the same conditions. For example, theplants grown from the seeds comprising the endophytic bacterialpopulation can have at least 5% higher WUE, for example, at least 10%higher, at least 20% higher, at least 30% higher, at least 40% higher,at least 50% higher, at least 60% higher, at least 70% higher, at least80% higher, at least 90% higher, at least 100% higher WUE than areference agricultural plant grown under the same conditions. Such anincrease in WUE can occur under conditions without water deficit, orunder conditions of water deficit, for example, when the soil watercontent is less than or equal to 60% of water saturated soil, forexample, less than or equal to 50%, less than or equal to 40%), lessthan or equal to 30%, less than or equal to 20%, less than or equal to10% of water saturated soil on a weight basis.

In a related embodiment, the plant comprising the endophytic bacterialendophyte can have at least 10%) higher relative water content (RWC),for example, at least 20% higher, at least 30% higher, at least 40%higher, at least 50% higher, at least 60% higher, at least 70% higher,at least 80%) higher, at least 90% higher, at least 100% higher RWC thana reference agricultural plant grown under the same conditions.

Many of the microbes described herein are capable of producing the planthormone auxin indole acetic acid (IAA) when grown in culture. Auxin mayplay a key role in altering the physiology of the plant, including theextent of root growth. Therefore, in another embodiment, the endophyticbacterial population is disposed on the surface or within a tissue ofthe seed or seedling in an amount effective to detectably induceproduction of auxin in the agricultural plant. For example, the increasein auxin production can be at least 10%, for example, at least 20%, atleast 30%), at least 40%, at least 50%, at least 60%, at least 75%, atleast 100%, or more, when compared with a reference agricultural plant.In one embodiment, the increased auxin production can be detected in atissue type selected from the group consisting of the root, shoot,leaves, and flowers.

In another embodiment, the endophytic bacterial population of thepresent invention can cause a detectable modulation in the amount of ametabolite in the plant or part of the plant. Such modulation can bedetected, for example, by measuring the levels of a given metabolite andcomparing with the levels of the metabolite in a reference agriculturalplant grown under the same conditions.

Plants Useful for the Present Invention

The methods and compositions according to the present invention can bedeployed for any seed plant species. Monocotyledonous as well asdicotyledonous plant species are particularly suitable. The methods andcompositions are preferably used with plants that are important orinteresting for agriculture, horticulture, for the production of biomassused in producing liquid fuel molecules and other chemicals, and/orforestry.

Thus, the invention has use over a broad range of plants, preferablyhigher plants pertaining to the classes of Angiospermae andGymnospermae. Plants of the subclasses of the Dicotylodenae and theMonocotyledonae are particularly suitable. Dicotyledonous plants belongto the orders of the Aristochiales, Asterales, Batales, Campanulales,Capparales, Caryophyllales, Casuarinales, Celastrales, Cornales,Diapensales, Dilleniales, Dipsacales, Ebenales, Ericales, Eucomiales,Euphorbiales, Fabales, Fagales, Gentianales, Geraniales, Haloragales,Hamamelidales, Middles, Juglandales, Lamiales, Laurales, Lecythidales,Leitneriales, Magniolales, Malvales, Myricales, Myrtales, Nymphaeales,Papeverales, Piperales, Plantaginales, Plumb aginales, Podostemales,Polemoniales, Polygalales, Polygonales, Primulales, Proteales,Rafflesiales, Ranunculales, Rhamnales, Rosales, Rubiales, Salicales,Santales, Sapindales, Sarraceniaceae, Scrophulariales, Theales,Trochodendrales, Umbellales, Urticales, and Violates. Monocotyledonousplants belong to the orders of the Alismatales, Arales, Arecales,Bromeliales, Commelinales, Cyclanthales, Cyperales, Eriocaulales,Hydrocharitales, Juncales, Lilliales, Najadales, Orchidales, Pandanales,Poales, Restionales, Triuridales, Typhales, and Zingiberales. Plantsbelonging to the class of the Gymnospermae are Cycadales, Ginkgoales,Gnetales, and Pinales.

Suitable species may include members of the genus Abelmoschus, Abies,Acer, Agrostis, Allium, Alstroemeria, Ananas, Andrographis, Andropogon,Artemisia, Arundo, Atropa, Berberis, Beta, Bixa, Brassica, Calendula,Camellia, Camptotheca, Cannabis, Capsicum, Carthamus, Catharanthus,Cephalotaxus, Chrysanthemum, Cinchona, Citrullus, Coffea, Colchicum,Coleus, Cucumis, Cucurbita, Cynodon, Datura, Dianthus, Digitalis,Dioscorea, Elaeis, Ephedra, Erianthus, Erythroxylum, Eucalyptus,Festuca, Fragaria, Galanthus, Glycine, Gossypium, Helianthus, Hevea,Hordeum, Hyoscyamus, Jatropha, Lactuca, Linum, Lolium, Lupinus,Lycopersicon, Lycopodium, Manihot, Medicago, Mentha, Miscanthus, Musa,Nicotiana, Oryza, Panicum, Papaver, Parthenium, Pennisetum, Petunia,Phalaris, Phleum, Pinus, Poa, Poinsettia, Populus, Rauwolfia, Ricinus,Rosa, Saccharum, Salix, Sanguinaria, Scopolia, Secale, Solanum, Sorghum,Spartina, Spinacea, Tanacetum, Taxus, Theobroma, Triticosecale,Triticum, Uniola, Veratrum, Vinca, Vitis, and Zea.

The methods and compositions of the present invention are preferablyused in plants that are important or interesting for agriculture,horticulture, biomass for the production of biofuel molecules and otherchemicals, and/or forestry. Non-limiting examples include, for instance,Panicum virgatum (switchgrass), Sorghum bicolor (sorghum, sudangrass),Miscanthus giganteus (miscanthus), Saccharum sp. (energycane), Populusbalsamifera (poplar), Zea mays (corn), Glycine max (soybean), Brassicanapus (canola), Triticum aestivum (wheat), Gossypium hirsutum (cotton),Oryza sativa (rice), Helianthus annuus (sunflower), Medicago sativa(alfalfa), Beta vulgaris (sugarbeet), Pennisetum glaucum (pearl millet),Panicum spp., Sorghum spp., Miscanthus spp., Saccharum spp., Erianthusspp., Populus spp., Secale cereale (rye), Salix spp. (willow),Eucalyptus spp. (eucalyptus), Triticosecale spp. (triticum-wheat X rye),Bamboo, Carthamus tinctorius (saffiower), Jatropha curcas (Jatropha),Ricinus communis (castor), Elaeis guineensis (oil palm), Phoenixdactylifera (date palm), Archontophoenix cunninghamiana (king palm),Syagrus romanzoffiana (queen palm), Linum usitatissimum (flax), Brassicajuncea, Manihot esculenta (cassava), Lycopersicon esculentum (tomato),Lactuca saliva (lettuce), Musa paradisiaca (banana), Solanum tuberosum(potato), Brassica oleracea (broccoli, cauliflower, brusselsprouts),Camellia sinensis (tea), Fragaria ananassa (strawberry), Theobroma cacao(cocoa), Coffea arabica (coffee), Vitis vinifera (grape), Ananas comosus(pineapple), Capsicum annum (hot & sweet pepper), Allium cepa (onion),Cucumis melo (melon), Cucumis sativus (cucumber), Cucurbita maxima(squash), Cucurbita moschata (squash), Spinacea oleracea (spinach),Citrullus lanatus (watermelon), Abelmoschus esculentus (okra), Solanummelongena (eggplant), Papaver somniferum (opium poppy), Papaverorientale, Taxus baccata, Taxus brevifolia, Artemisia annua, Cannabissaliva, Camptotheca acuminate, Catharanthus roseus, Vinca rosea,Cinchona officinalis, Coichicum autumnale, Veratrum californica,Digitalis lanata, Digitalis purpurea, Dioscorea spp., Andrographispaniculata, Atropa belladonna, Datura stomonium, Berberis spp.,Cephalotaxus spp., Ephedra sinica, Ephedra spp., Erythroxylum coca,Galanthus wornorii, Scopolia spp., Lycopodium serratum (Huperziaserrata), Lycopodium spp., Rauwolfia serpentina, Rauwolfia spp.,Sanguinaria canadensis, Hyoscyamus spp., Calendula officinalis,Chrysanthemum parthenium, Coleus forskohlii, Tanacetum parthenium,Parthenium argentatum (guayule), Hevea spp. (rubber), Mentha spicata(mint), Mentha piperita (mint), Bixa orellana, Alstroemeria spp., Rosaspp. (rose), Dianthus caryophyllus (carnation), Petunia spp. (petunia),Poinsettia pulcherrima (Poinsettia), Nicotiana tabacum (tobacco),Lupinus albus (lupin), Uniola paniculata (oats), Hordeum vulgare(barley), and Lolium spp. (ryegrass).

The methods described herein can also be used with genetically modifiedplants, for example, to yield additional trait benefits to a plant. Forexample, a genetically modified plant which is, by means of thetransgene, optimized with respect to a certain trait, can be furtheraugmented with additional trait benefits conferred by the newlyintroduced microbe. Therefore, in one embodiment, a genetically modifiedplant is contacted with a microbe.

Formulations/Seed Coating Compositions

In some embodiments, the present invention contemplates seeds comprisinga endophytic bacterial population, and further comprising a formulation.The formulation useful for these embodiments generally comprise at leastone member selected from the group consisting of an agriculturallycompatible carrier, a tackifier, a microbial stabilizer, a fungicide, anantibacterial agent, an herbicide, a nematicide, an insecticide, a plantgrowth regulator, a rodenticide, and a nutrient.

In some cases, the endophytic bacterial population is mixed with anagriculturally compatible carrier. The carrier can be a solid carrier orliquid carrier. The carrier may be any one or more of a number ofcarriers that confer a variety of properties, such as increasedstability, wettability, or dispersability. Wetting agents such asnatural or synthetic surfactants, which can be nonionic or ionicsurfactants, or a combination thereof can be included in a compositionof the invention. Water-in-oil emulsions can also be used to formulate acomposition that includes the endophytic bacterial population of thepresent invention (see, for example, U.S. Pat. No. 7,485,451, which isincorporated herein by reference in its entirety). Suitable formulationsthat may be prepared include wettable powders, granules, gels, agarstrips or pellets, thickeners, and the like, microencapsulatedparticles, and the like, liquids such as aqueous flowables, aqueoussuspensions, water-in-oil emulsions, etc. The formulation may includegrain or legume products, for example, ground grain or beans, broth orflour derived from grain or beans, starch, sugar, or oil.

In some embodiments, the agricultural carrier may be soil or plantgrowth medium. Other agricultural carriers that may be used includefertilizers, plant-based oils, humectants, or combinations thereof.Alternatively, the agricultural carrier may be a solid, such asdiatomaceous earth, loam, silica, alginate, clay, bentonite,vermiculite, seed cases, other plant and animal products, orcombinations, including granules, pellets, or suspensions. Mixtures ofany of the aforementioned ingredients are also contemplated as carriers,such as but not limited to, pesta (flour and kaolin clay), agar orflour-based pellets in loam, sand, or clay, etc. Formulations mayinclude food sources for the cultured organisms, such as barley, rice,or other biological materials such as seed, plant parts, sugar canebagasse, hulls or stalks from grain processing, ground plant material orwood from building site refuse, sawdust or small fibers from recyclingof paper, fabric, or wood. Other suitable formulations will be known tothose skilled in the art.

In one embodiment, the formulation can comprise a tackifier or adherent.Such agents are useful for combining the bacterial population of theinvention with carriers that can contain other compounds (e.g., controlagents that are not biologic), to yield a coating composition. Suchcompositions help create coatings around the plant or seed to maintaincontact between the microbe and other agents with the plant or plantpart. In one embodiment, adherents are selected from the groupconsisting of: alginate, gums, starches, lecithins, formononetin,polyvinyl alcohol, alkali formononetinate, hesperetin, polyvinylacetate, cephalins, Gum Arabic, Xanthan Gum, Mineral Oil, PolyethyleneGlycol (PEG), Polyvinyl pyrrolidone (PVP), Arabino-galactan, MethylCellulose, PEG 400, Chitosan, Polyacrylamide, Polyacrylate,Polyacrylonitrile, Glycerol, Triethylene glycol, Vinyl Acetate, GellanGum, Polystyrene, Polyvinyl, Carboxymethyl cellulose, Gum Ghatti, andpolyoxyethylene-polyoxybutylene block copolymers. Other examples ofadherent compositions that can be used in the synthetic preparationinclude those described in EP 0818135, CA 1229497, WO 2013090628, EP0192342, WO 2008103422 and CA 1041788, each of which is incorporatedherein by reference in its entirety.

The formulation can also contain a surfactant. Non-limiting examples ofsurfactants include nitrogen-surfactant blends such as Prefer 28(Cenex), Surf-N(US), Inhance (Brandt), P-28 (Wilfarm) and Patrol(Helena); esterified seed oils include Sun-It II (AmCy), MSO (UAP),Scoil (Agsco), Hasten (Wilfarm) and Mes-100 (Drexel); andorgano-silicone surfactants include Silwet L77 (UAP), Silikin (Terra),Dyne-Amic (Helena), Kinetic (Helena), Sylgard 309 (Wilbur-Ellis) andCentury (Precision). In one embodiment, the surfactant is present at aconcentration of between 0.01% v/v to 10%) v/v. In another embodiment,the surfactant is present at a concentration of between 0.1% v/v to 1%v/v.

In certain cases, the formulation includes a microbial stabilizer. Suchan agent can include a desiccant. As used herein, a “desiccant” caninclude any compound or mixture of compounds that can be classified as adesiccant regardless of whether the compound or compounds are used insuch concentrations that they in fact have a desiccating effect on theliquid inoculant. Such desiccants are ideally compatible with thebacterial population used, and should promote the ability of themicrobial population to survive application on the seeds and to survivedesiccation. Examples of suitable desiccants include one or more oftrehalose, sucrose, glycerol, and methylene glycol. Other suitabledesiccants include, but are not limited to, non reducing sugars andsugar alcohols (e.g., mannitol or sorbitol). The amount of desiccantintroduced into the formulation can range from about 5% to about 50% byweight/volume, for example, between about 10% to about 40%, betweenabout 15%) and about 35%, or between about 20% and about 30%.

In some cases, it is advantageous for the formulation to contain agentssuch as a fungicide, an antibacterial agent, an herbicide, a nematicide,an insecticide, a plant growth regulator, a rodenticide, and a nutrient.Such agents are ideally compatible with the agricultural seed orseedling onto which the formulation is applied (e.g., it should not bedeleterious to the growth or health of the plant). Furthermore, theagent is ideally one which does not cause safety concerns for human,animal or industrial use (e.g., no safety issues, or the compound issufficiently labile that the commodity plant product derived from theplant contains negligible amounts of the compound).

In the liquid form, for example, solutions or suspensions, theendophytic bacterial populations of the present invention can be mixedor suspended in aqueous solutions. Suitable liquid diluents or carriersinclude aqueous solutions, petroleum distillates, or other liquidcarriers.

Solid compositions can be prepared by dispersing the endophyticbacterial populations of the invention in and on an appropriatelydivided solid carrier, such as peat, wheat, bran, vermiculite, clay,talc, bentonite, diatomaceous earth, fuller's earth, pasteurized soil,and the like. When such formulations are used as wettable powders,biologically compatible dispersing agents such as non-ionic, anionic,amphoteric, or cationic dispersing and emulsifying agents can be used.

The solid carriers used upon formulation include, for example, mineralcarriers such as kaolin clay, pyrophyllite, bentonite, montmorillonite,diatomaceous earth, acid white soil, vermiculite, and pearlite, andinorganic salts such as ammonium sulfate, ammonium phosphate, ammoniumnitrate, urea, ammonium chloride, and calcium carbonate. Also, organicfine powders such as wheat flour, wheat bran, and rice bran may be used.The liquid carriers include vegetable oils such as soybean oil andcottonseed oil, glycerol, ethylene glycol, polyethylene glycol,propylene glycol, polypropylene glycol, etc.

In one particular embodiment, the formulation is ideally suited forcoating of the endophytic microbial population onto seeds. Theendophytic bacterial populations described in the present invention arecapable of conferring many fitness benefits to the host plants. Theability to confer such benefits by coating the bacterial populations onthe surface of seeds has many potential advantages, particularly whenused in a commercial (agricultural) scale.

The endophytic bacterial populations herein can be combined with one ormore of the agents described above to yield a formulation suitable forcombining with an agricultural seed or seedling. The bacterialpopulation can be obtained from growth in culture, for example, using asynthetic growth medium. In addition, the microbe can be cultured onsolid media, for example on petri dishes, scraped off and suspended intothe preparation. Microbes at different growth phases can be used. Forexample, microbes at lag phase, early-log phase, mid-log phase, late-logphase, stationary phase, early death phase, or death phase can be used.

The formulations comprising the endophytic bacterial population of thepresent invention typically contains between about 0.1 to 95% by weight,for example, between about 1% and 90%, between about 3% and 75%, betweenabout 5% and 60%, between about 10% and 50% in wet weight of thebacterial population of the present invention. It is preferred that theformulation contains at least about 10³ per ml of formulation, forexample, at least about 10⁴, at least about 10⁵, at least about 10⁶, atleast 10⁷ CFU, at least 10⁸ CFU per ml of formulation.

As described above, in certain embodiments, the present inventioncontemplates the use of endophytic bacteria that are heterologouslydisposed on the plant, for example, the seed. In certain cases, theagricultural plant may contain bacteria that are substantially similarto, or even genetically indistinguishable from, the bacteria that arebeing applied to the plant. It is noted that, in many cases, thebacteria that are being applied is substantially different from thebacteria already present in several significant ways. First, thebacteria that are being applied to the agricultural plant have beenadapted to culture, or adapted to be able to grow on growth media inisolation from the plant. Second, in many cases, the bacteria that arebeing applied are derived from a clonal origin, rather than from aheterologous origin and, as such, can be distinguished from the bacteriathat are already present in the agricultural plant by the clonalsimilarity. For example, where a microbe that has been inoculated by aplant is also present in the plant (for example, in a different tissueor portion of the plant), or where the introduced microbe issufficiently similar to a microbe that is present in some of the plants(or portion of the plant, including seeds), it is still possible todistinguish between the inoculated microbe and the native microbe bydistinguishing between the two microbe types on the basis of theirepigenetic status (e.g., the bacteria that are applied, as well as theirprogeny, would be expected to have a much more uniform and similarpattern of cytosine methylation of its genome, with respect to theextent and/or location of methylation).

Population of Seeds

In another aspect, the invention provides for a substantially uniformpopulation of seeds comprising a plurality of seeds comprising theendophytic bacterial population, as described herein above. Substantialuniformity can be determined in many ways. In some cases, at least 10%,for example, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 75%), at least 80%, at least 90%, atleast 95% or more of the seeds in the population, contains theendophytic bacterial population in an amount effective to colonize theplant disposed on the surface of the seeds. In other cases, at least10%, for example, at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 75%, at least 80%, at least90%, at least 95% or more of the seeds in the population, contains atleast 100 CFU on its surface, for example, at least 200 CFU, at least300 CFU, at least 1,000 CFU, at least 3,000 CFU, at least 10,000 CFU, atleast 30,000 CFU, at least 100,000 CFU, at least 300,000 CFU, or atleast 1,000,000 CFU per seed or more.

In a particular embodiment, the population of seeds is packaged in a bagor container suitable for commercial sale. Such a bag contains a unitweight or count of the seeds comprising the endophytic bacterialpopulation as described herein, and further comprises a label. In oneembodiment, the bag or container contains at least 1,000 seeds, forexample, at least 5,000 seeds, at least 10,000 seeds, at least 20,000seeds, at least 30,000 seeds, at least 50,000 seeds, at least 70,000seeds, at least 80,000 seeds, at least 90,000 seeds or more. In anotherembodiment, the bag or container can comprise a discrete weight ofseeds, for example, at least 1 lb, at least 2 lbs, at least 5 lbs, atleast 10 lbs, at least 30 lbs, at least 50 lbs, at least 70 lbs or more.The bag or container comprises a label describing the seeds and/or saidendophytic bacterial population. The label can contain additionalinformation, for example, the information selected from the groupconsisting of: net weight, lot number, geographic origin of the seeds,test date, germination rate, inert matter content, and the amount ofnoxious weeds, if any. Suitable containers or packages include thosetraditionally used in plant seed commercialization. The invention alsocontemplates other containers with more sophisticated storagecapabilities (e.g., with microbiologically tight wrappings or with gas-or water-proof containments).

In some cases, a sub-population of seeds comprising the endophyticbacterial population is further selected on the basis of increaseduniformity, for example, on the basis of uniformity of microbialpopulation. For example, individual seeds of pools collected fromindividual cobs, individual plants, individual plots (representingplants inoculated on the same day) or individual fields can be testedfor uniformity of microbial density, and only those pools meetingspecifications (e.g., at least 80% of tested seeds have minimum density,as determined by quantitative methods described elsewhere) are combinedto provide the agricultural seed sub-population.

The methods described herein can also comprise a validating step. Thevalidating step can entail, for example, growing some seeds collectedfrom the inoculated plants into mature agricultural plants, and testingthose individual plants for uniformity. Such validating step can beperformed on individual seeds collected from cobs, individual plants,individual plots (representing plants inoculated on the same day) orindividual fields, and tested as described above to identify poolsmeeting the required specifications.

Population of Plants/Agricultural Fields

A major focus of crop improvement efforts has been to select varietieswith traits that give, in addition to the highest return, the greatesthomogeneity and uniformity. While inbreeding can yield plants withsubstantial genetic identity, heterogeneity with respect to plantheight, flowering time, and time to seed, remain impediments toobtaining a homogeneous field of plants. The inevitable plant-to-plantvariability are caused by a multitude of factors, including unevenenvironmental conditions and management practices. Another possiblesource of variability can, in some cases, be due to the heterogeneity ofthe microbial population inhabit the plants. By providing endophyticbacterial populations onto seeds and seedlings, the resulting plantsgenerated by germinating the seeds and seedlings have a more consistentmicrobial composition, and thus are expected to yield a more uniformpopulation of plants.

Therefore, in another aspect, the invention provides a substantiallyuniform population of plants. The population comprises at least 100plants, for example, at least 300 plants, at least 1,000 plants, atleast 3,000 plants, at least 10,000 plants, at least 30,000 plants, atleast 100,000 plants or more. The plants are grown from the seedscomprising the endophytic bacterial population as described herein. Theincreased uniformity of the plants can be measured in a number ofdifferent ways.

In one embodiment, there is an increased uniformity with respect to themicrobes within the plant population. For example, in one embodiment, asubstantial portion of the population of plants, for example at least10%, at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 70%), at least 75%, at least 80%, at least 90%, at least95% or more of the seeds or plants in a population, contains a thresholdnumber of the endophytic bacterial population. The threshold number canbe at least 100 CFU, for example at least 300 CFU, at least 1,000 CFU,at least 3,000 CFU, at least 10,000 CFU, at least 30,000 CFU, at least100,000 CFU or more, in the plant or a part of the plant. Alternatively,in a substantial portion of the population of plants, for example, in atleast 1%, at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 75%), at least 80%, atleast 90%, at least 95% or more of the plants in the population, theendophytic bacterial population that is provided to the seed or seedlingrepresents at least 10%, least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 99%, or 100% of the total microbe population in theplant/seed.

In another embodiment, there is an increased uniformity with respect toa physiological parameter of the plants within the population. In somecases, there can be an increased uniformity in the height of the plantswhen compared with a population of reference agricultural plants grownunder the same conditions. For example, there can be a reduction in thestandard deviation in the height of the plants in the population of atleast 5%, for example, at least 10%, at least 15%, at least 20%), atleast 30%, at least 40%, at least 50%, at least 60% or more, whencompared with a population of reference agricultural plants grown underthe same conditions. In other cases, there can be a reduction in thestandard deviation in the flowering time of the plants in the populationof at least 5%>, for example, at least 10%, at least 15%, at least 20%,at least 30%, at least 40%, at least 50%), at least 60% or more, whencompared with a population of reference agricultural plants grown underthe same conditions.

Commodity Plant Product

The present invention provides a commodity plant product, as well asmethods for producing a commodity plant product, that is derived from aplant of the present invention. As used herein, a “commodity plantproduct” refers to any composition or product that is comprised ofmaterial derived from a plant, seed, plant cell, or plant part of thepresent invention. Commodity plant products may be sold to consumers andcan be viable or nonviable. Nonviable commodity products include but arenot limited to nonviable seeds and grains; processed seeds, seed parts,and plant parts; dehydrated plant tissue, frozen plant tissue, andprocessed plant tissue; seeds and plant parts processed for animal feedfor terrestrial and/or aquatic animal consumption, oil, meal, flour,flakes, bran, fiber, paper, tea, coffee, silage, crushed of whole grain,and any other food for human or animal consumption; and biomasses andfuel products; and raw material in industry. Industrial uses of oilsderived from the agricultural plants described herein includeingredients for paints, plastics, fibers, detergents, cosmetics,lubricants, and biodiesel fuel. Soybean oil may be split,inter-esterified, sulfurized, epoxidized, polymerized, ethoxylated, orcleaved. Designing and producing soybean oil derivatives with improvedfunctionality and improved oliochemistry is a rapidly growing field. Thetypical mixture of triglycerides is usually split and separated intopure fatty acids, which are then combined with petroleum-derivedalcohols or acids, nitrogen, sulfonates, chlorine, or with fattyalcohols derived from fats and oils to produce the desired type of oilor fat. Commodity plant products also include industrial compounds, suchas a wide variety of resins used in the formulation of adhesives, films,plastics, paints, coatings and foams.

In some cases, commodity plant products derived from the plants, orusing the methods of the present invention can be identified readily. Insome cases, for example, the presence of viable endophytic microbes canbe detected using the methods described herein elsewhere. In othercases, particularly where there are no viable endophytic microbes, thecommodity plant product may still contain at least a detectable amountof the specific and unique DNA corresponding to the microbes describedherein. Any standard method of detection for polynucleotide moleculesmay be used, including methods of detection disclosed herein.

Throughout the specification, the word “comprise,” or variations such as“comprises” or “comprising,” will be understood to imply the inclusionof a stated integer or group of integers but not the exclusion of anyother integer or group of integers.

Although the present invention has been described in detail withreference to examples below, it is understood that various modificationscan be made without departing from the spirit of the invention. Forinstance, while the particular examples below may illustrate the methodsand embodiments described herein using a specific plant, the principlesin these examples may be applied to any agricultural crop. Therefore, itwill be appreciated that the scope of this invention is encompassed bythe embodiments of the inventions recited herein and the specificationrather than the specific examples that are exemplified below. All citedpatents and publications referred to in this application are hereinincorporated by reference in their entirety.

EXAMPLES Example 1: Phenotypic and Physiological Characterization ofMicrobes

Bacterial strains from overnight cultures in tryptic soy broth werestreaked on tryptic soy agar (TSA) plates and incubated at 30° C. After24 h, the color and shape of colonies were noted. Cell motility andcolony shape were observed under light microscope (Nikon, Japan). The pHlimits for bacterial growth was determined by adjusting the pH of thegrowth media to values between 5 and 12 in triplicates. Bacterial growthon different salt concentrations was tested in TSA medium containing1-6% NaCl. Furthermore, the ability of the microbes to grow inmethanol/ethanol as sole carbon source was analyzed by replacing theglucose with either methanol or ethanol.

Aggregate formation of bacterial strains can positively affect theirdispersal and survival in the plant environment and adsorption to plantroots. The extent of aggregation formation was measured in sixreplicates following the method of Madi and Henis (1989) Plant Soil115:89-98 (incorporated herein by reference) with some modifications.Aliquots of liquid culture containing aggregates were transferred toglass tubes and allowed to stand for 30 min. Aggregates settled down tothe bottom of each tubes, and the suspension was mostly composed free ofcells. The turbidity of each suspension was measured at 540 nm (ODs)with a microplate reader (Synergy 5; BioTek Instrument Inc., Winooski,USA). Cultures were then dispersed with a tissue homogenizer for 1 minand the total turbidity (OD) was measured. The percentage of aggregationwas estimated as follows:% aggregation=(ODt−ODs)×100/ODt

Motility assays (swimming, swarming and twitching) were performedfollowing the methods of Rashid and Kornberg (2000). Swim plates (LBmedia contained 0.3% agarose) were inoculated in triplicates withbacteria from an overnight culture on TSA agar plates grown at 30° C.with a sterile toothpick. For swarming, plates (NB media contained 0.5%agar and glucose) were inoculated with a sterile toothpick. Twitchplates (LB broth containing 1% Difco granular agar) were stab inoculatedwith a sharp toothpick to the bottom of petri dish from an overnightgrown culture in TSA agar plates.

Biofilm formation was analyzed using overnight grown bacterial culturein 96 well microtiter plates by staining with 1% crystal violet (CV) for45 min. To quantify the amount of biofilm, CV was destained with 200 μlof 100% ethanol. The absorbance of 150 μl of the destained CV, which wastransferred into a new microtiter plate was measured at 595 nm (modifiedfrom Djordjevic et al. 2002, Appl Environ Microbiol 68:2950-2958,incorporated herein by reference).

The Phenotypic Characteristics of the Strains are Shown Below in Table1:

Agrobacterium Pantoea Sphinogobium Pseudomonus Enterobacter MicrococusCharacteristics sp. (FA13) sp. (FF34) sp. (FC42) sp. (FB12) sp. (FD17)sp. S2 Phenotypic and physiological characterization Colony color GrayYellow Yellow Gray Creamy Creamy white Colonymorphology Round RoundRound Round Round Round Gram reaction n.d. n.d. n.d. n.d. n.d. positiveBacterial growth conditions* Temperature  4° C. n.d. n.d. n.d. n.d. n.d.− 42° C. n.d. n.d. n.d. n.d. n.d. − NaCl 2% + + + + + + 6% − + − − + +pH  5 + + + + + + 12 + − − + + + Motility/chemotaxis Swimming + + − +++++ − Swarming − − − − + − Twitching + + − + + − Biofilm formation OD(600 nm) 0.92 ± 0.04 0.59 ± 0.02 0.95 ± 0.08 0.57 ± 0.08 0.95 ± 0.040.92 ± 0.04 Biofilm (595 nm) 0.23 ± 0.02 0.22 ± 0.03 0.08 ± 0.01 0.08 ±0.04 0.83 ± 0.06 0.23 ± 0.02 Aggregate 35.91 ± 2.57  26.07 ± 0.88  32.61± 2.13  36.38 ± 1.48  40.22 ± 1.99  35.91 ± 2.57  stability (%) BacillusPantoea Acinetobacter Paenibacillus Characteristics sp. S4 sp. S6 sp. S9sp. S10 Phenotypic and physiological characterization Colony color Off-Yellow White Creamy white white Colonymorphology Round Round Round RoundGram reaction positive negative negative negative Bacterial growthconditions* Temperature  4° C. + + + + 42° C. − − − − NaCl 2% + + + +6% + + − + pH  5 + + + + 12 − + − + Motility/chemotaxis Swimming − + −++ Swarming − ++ − + Twitching + + − + Biofilm formation OD (600 nm)0.59 ± 0.02 0.95 ± 0.08 0.57 ± 0.08 0.95 ± 0.04 Biofilm (595 nm) 0.22 ±0.03 0.08 ± 0.01 0.08 ± 0.04 0.83 ± 0.06 Aggregate 26.07 ± 0.88  32.61 ±2.13  36.38 ± 1.48  40.22 ± 1.99  stability (%)Biochemical Characterization

Biochemical tests such as oxidase, catalase, gelatin hydrolysis andcasein hydrolysis of the selected strains were performed. Oxidase andcatalase activities were tested with 1% (w/v) tetramethyl-p-phenylenediamine and 3% (v/v) hydrogen peroxide solution, respectively. Gelatinand casein hydrolysis was performed by streaking bacterial strains ontoa TSA plates from the stock culture. After incubation, trichloroaceticacid (TCA) was applied to the plates and made observation immediatelyfor a period of at least 4 min (Medina and Baresi 2007, J MicrobiolMethods 69:391-393, incorporated herein by reference).

A Summary of the Biochemical Characteristics of the Strains is ShownBelow in Table 2:

TABLE 2 Biochemical Characterization of Endophytic Bacteria Biochemicalcharacterization* Agrobacterium Pantoea Sphinogobium PseudomonasEnterobacter Micrococus sp. (FA13) sp. (FF34) sp. (FC42) sp. (FBI 2) sp.(FD17) sp. S2 Catalase + + + + + + Oxidase − − − + − − Casein − − − +− + Gelatin − + − + + + Methanol + − − + − + Ethanol + − − + − +Bacillus Pantoea Acinetobacter Paenibacillus sp. S4 sp. S6 sp. S9 sp.S10 Catalase + + + + Oxidase + − − + Casein + − − − Gelatin − + − −Methanol − − + + Ethanol − − + +Quantification of Auxin Production

Auxin production by bacterial isolates both in the presence and absenceof L-tryptophan (L-TRP) was determined colormetrically and expressed asIAA equivalent (Sarwar et al. 1992, Plant Soil 147:207-215, incorporatedherein by reference). Two days old bacterial cells grown (28° C. at 180rpm) in tryptic soy broth supplemented with 1% L-TRP solution wereharvested by centrifugation (10,000 g for 10 min). Three mL of thesupernatants were mixed with 2 mL Salkowski's reagent (12 g L⁻¹ FeC13 in429 ml L⁻¹ H2504). The mixture was incubated at room temperature for 30min for colour development and absorbance at 535 nm was measured usingspectrophotometer. Auxin concentration produced by bacterial isolateswas determined using standard curves for IAA prepared from serialdilutions of 10-100 μg mL⁻¹.

TABLE 3 Production of Indole Acetic Acid by Endophytic BacteriaAgrobacterium sp. Pantoea sp. Sphinogobium sp. Pseudomonas sp.Enterobacter sp. Characteristics (FA13) (FF34) (FC42) (FBI 2) (FD17)without L-TRP  1.74 ± 0.18 10.33 ± 0.35  4.89 ± 0.78 1.63 ± 0.65  7.54 ±1.02 with L-TRP 16.13 ± 1.05 95.34 ± 2.14 38.41 ± 1.78 7.26 ± 1.05 12.30± 0.98

As shown in Table 3 above, strains FA13, FF34, FC42, FB12 and FD17 wereall shown to produce auxin (ranging from 1.63 to 10.33 μg ml⁻¹ in theabsence of L-tryptophan), and the level of auxin production was greatlyenhanced by the presence of L-tryptophan in the growth medium (at least7.26 μg ml⁻¹).

Assays for Phosphorus Solubilization and Siderophore Production

Bacterial strains were evaluated for their ability to solubilizephosphates (organic/inorganic P). Aliquots (10 μL) of overnightbacterial growth culture in tryptic soy broth were spot inoculated ontoNBRI-PBP (Mehta and Nautiyal 2001) and calcium/sodium phytate agarmedium (Rosado et al. 1998). Solubilization of organic/inorganicphosphates was detected by the formation of a clear zone around thebacterial growth spot. Phosphate solubilization activity was alsodetermined by development of clear zone around bacterial growth onPikovskaya agar medium (Pikovskaya 1948, Mikrobiologiya 17:362-370,incorporated herein by reference). Bacterial isolates were assayed forsiderophores production on the Chrome azurol S (CAS) agar mediumdescribed by Schwyn and Neilands (1987), Curr Microbiol 43:57-58(incorporated herein by reference) as positive for siderophoreproduction.

Assays for Exopolysaccharide, NH₃ and HCN Production

For exopolysaccharide (EPS) activity (qualitative), strains were grownon Weaver mineral media enriched with glucose and production of EPS wasassessed visually (modified from Weaver et al. 1975, Arch Microbiol105:207-216, incorporated herein by reference). The EPS production wasmonitored as floe formation (fluffy material) on the plates after 48 hof incubation at 28±2° C. Strains were tested for the production ofammonia (NH₃) in peptone water as described by Cappuccino and Sherman(1992), Biochemical activities of microorganisms. In: Microbiology, ALaboratory Manual. The Benjamin/Cummings Publishing Co. California, USA,pp 125-178, incorporated herein by reference. The bacterial isolateswere screened for the production of hydrogen cyanide (HCN) byinoculating King's B agar plates amended with 4.4 g L⁻¹ glycine (Lorck1948, Physiol Plant 1:142-146, incorporated herein by reference). Filterpaper (Whatman no. 1) saturated with picrate solution (2% Na₂CO₃ in 0.5%picric acid) was placed in the lid of a petri plate inoculated withbacterial isolates. The plates were incubated at 28±2° C. for 5 days.HCN production was assessed by the colour change of yellow filter paperto reddish brown.

Assays for Poly-Hydroxybutyrate (PHB) and n-Acyl-Homoserine Lactone(AHL) Production

The bacterial isolates were tested for PHB production (qualitative)following the viable colony staining methods using Nile red and Sudanblack B (Juan et al. 1998 Appl Environ Microbiol 64:4600-4602;Spiekermann et al. 1999, Arch Microbiol 171:73-80, each of which isincorporated by reference). The LB plates with overnight bacterialgrowth were flooded with 0.02% Sudan black B for 30 min and then washedwith ethanol (96%) to remove excess strains from the colonies. The darkblue coloured colonies were taken as positive for PHB production.Similarly, LB plates amended with Nile red (0.5 μL mL⁻¹) were exposed toUV light (312 nm) after appropriate bacterial growth to detect PHBproduction. Colonies of PHA-accumulating strains showed fluoresce underultraviolet light. The bacterial strains were tested for AHL productionfollowing the method modified from Cha et al. (1998), Mol Plant-MicrobeInteract 11:1119-1129 (incorporated herein by reference). The LB platescontaining 40 ug ml⁻¹ X-Gal were plated with reporter strains (A.tumefaciens NTL4.pZLR4). The LB plates were spot inoculated with 10 μLof bacterial culture and incubated at 28±2° C. for 24 h. Production ofAHL activity is indicated by a diffuse blue zone surrounding the testspot of culture. Agrobacterium tumefaciens NTL1 (pTiC58ΔaccR) was usedas positive control and plate without reporter strain was considered asnegative control.

TABLE 4 Various Biochemical Properties of Endophytic BacteriaAgrobacterium Pantoea Sphinogobium Pseudomonas Enterobacter MicrococusCharacteristics sp. (FA13) sp. (FF34) sp. (FC42) sp. (FBI 2) sp. (FD17)sp. S2 P-solubilization (inorganic/organic P) Ca₃(PO₄)₂ − ++ − + +++ −CaHPCU − ++ − + +++ − Ca-phytate − ++ − ++ +++ − Na-phytate − ++ − +++++ − Exopoly ++ + + saccharide N₂-fixation + + − − + − HCN + productionNH₃ + + + + + + production Siderophore +++ + + ++ +++ n.d. productionAHL − − − + − − PHB − + − + + + Bacillus Pantoea AcinetobacterPaenibacillus Characteristics sp. S4 sp. S6 sp. S9 sp. S10P-solubilization (inorganic/organic P) Ca₃(PO₄)₂ − + − + CaHPCU − + − +Ca-phytate − + − + Na-phytate − + − + Exopoly + saccharide N₂-fixation− + − − HCN production NH₃ + + + + production Siderophore n.d. +production AHL − + − + PHB − + − −

As shown above, the bacteria described herein exhibit varying degrees ofphosphate utilization. For example, strains FF34, FB12, FD17, S6, andS10 were capable of hydrolyzing Ca₃(PO₄)₂, CaHPO₄, Ca-phytate andNa-phytate. These strains, therefore, may be effective for increasingphosphate availability for host plants under conditions of limitingconcentrations of soluble phosphate in the soil.

Siderophores are small, high-affinity iron chelating compounds secretedby microorganisms such as bacteria, fungi and grasses, siderophores.They bind to the available form of iron Fe³⁺ in the rhizosphere, thusmaking it unavailable to the phytopathogens and protecting the planthealth (Ahmad et al. 2008, Microbiol Res 163:173-181, incorporatedherein by reference). Siderophores are known for mobilizing Fe andmaking it available to the plant. Several of the strains, includingFA13, FF34, FC42, FB12, FD17 and S10 were found to produce significantlevels of siderophore when tested in agar medium containing Chrom azurolS (CAS). Therefore, in one embodiment, the strains described above areeffective in increasing iron availability to the host plant.

The ability of bacterial strains to utilize or metabolize differentnitrogen sources was evaluated. Interestingly, four of the strainstested (FA13, FF34, FD17, and S6) were capable of growing innitrogen-free medium, demonstrating their ability to fix nitrogen.Therefore, in one embodiment, these strains can be provided in an amounteffective to increase nitrogen utilization in a host plant.

Bacterial survival and colonization in the plant environment arenecessary for plant growth and yield. Recently, Zuniga and colleagues(2013), Mol Plant-Microbe Interact 26:546-553 (incorporated herein byreference) described that the cell-to-cell communication (QS) systemmediated by AHL is implicated in rhizosphere competence and colonizationof Arabidopsis thaliana by B. phytofirmans PsJN. Motility, aggregatestability, and biofilm formation are important traits for root surfacecolonization (Danhorn and Fuqua 2007, Annu Rev Microbiol 61:401-422,incorporated herein by reference). Three strains (FB12, S6 and S10) werefound to produce AHL. It should be noted, however, that the bacteriadescribed here may have other communication systems. Aggregation andbiofilm formation were common traits in all tested strains. In the caseof motility, six strains (FA13, FF34, FB12, FD17, S6 and S10) werepositive for swimming, while FD17, S6 and S10 also showed swarming.Therefore, in one embodiment, the seeds are provided with an amount ofthese strains in an amount effective to produce detectable levels ofAHL. In another embodiment, seeds of an agricultural plant are providedwith an amount of the bacterial endophyte population effective to formbiofilms.

Bacteria were tested for production of exopolysaccharide (EPS) andpoly-hydroxybutyrate (PHB). Bacterial EPS and PHB have been shown toprovide protection from such environmental insults as desiccation,predation, and the effects of antibiotics (Gasser et al. 2009, FEMSMicrobiol Ecol 70:142-150; Staudt et al. 2012, Arch Microbiol194:197-206, each of which is incorporated by reference). They can alsocontribute to bacterial aggregation, surface attachment, andplant-microbe symbiosis (Laus et al. 2005, Mol Plant-Microbe Interact18:533-538, incorporated herein by reference). Five strains (FF34, FB12,FD17, S2 and S6) showed PHB production, while FA13, FC42, FD17 and S10were found to produce EPS. Therefore, in another embodiment, seeds of anagricultural plant are provided with an amount of the bacterialendophyte population effective to improve desiccation tolerance in thehost plant.

Volatile compounds such as ammonia and HCN produced by a number ofrhizobacteria were reported to play an important role in biocontrol(Brimecombe et al. 2001, In: Pinton R, Varanini Z, Nannipieri P (Eds.)The Rhizosphere, Marcel Dekker, New York, pp 95-140, incorporated hereinby reference). Production of ammonia was commonly detected in allselected isolates. In contrast, only Pseudomonas sp. strain FB12 wasable to produce HCN. Among the strains tested, only FB12 was able toproduce HCN.

Enzyme Hydrolyzing Activities

Bacterial hydrolyzing activities due to amylase, cellulase, chitinase,hemolytic, lipase, pectinase, protease and xylanase were screened ondiagnostic plates after incubation at 28° C. Amylase activity wasdetermined on agar plates following the protocol Mannisto and Haggblom(2006), Syst Appl Microbiol 29:229-243, incorporated herein byreference. Formation of opaque halo around colonies was used as anindication of lipase activity. Cellulase and xylanase activities wereassayed on plates containing (per liter) 5 g of carboxymethyl celluloseor birch wood xylan, 1 g of peptone and 1 g of yeast extract. After 10days of incubation, the plates were flooded with gram's iodine stainingand washing with 1M NaCl to visualize the halo zone around the bacterialgrowth (modified from Teather and Wood 1982, Appl Environ Microbiol43:777-780, incorporated herein by reference). Chitinase activity of theisolates was determined as zones of clearing around colonies followingthe method of Chernin et al. (1998) J Bacteriol 180:4435-4441(incorporated herein by reference). Hemolytic activity was determined bystreaking bacterial isolates onto Columbia 5% sheep blood agar plates.Protease activity was determined using 1% skimmed milk agar plates,while lipase activity was determined on peptone agar medium. Formationof halo zone around colonies was used as indication of activity (Smibertand Krieg 1994, In: Gerhardt P, Murray R, Wood W, Krieg N (Eds) Methodsfor General and Molecular Bacteriology, ASM Press, Washington, D.C., pp615-640, incorporated herein by reference). Pectinase activity wasdetermined on nutrient agar supplemented with 5 g L⁻¹ pectin. After 1week of incubation, plates were flooded with 2% hexadecyl trimethylammonium bromide solution for 30 min. The plates were washed with 1MNaCl to visualize the halo zone around the bacterial growth (Mateos etal. 1992, Appl Environ Microbiol 58:1816-1822, incorporated herein byreference).

TABLE 5 Enzyme Activities from Endophytic Bacteria Agrobacterium PantoeaSphinogobium Pseudomonas Enterobacter Micrococus Characteristics sp.(FA13) sp. (FF34) sp. (FC42) sp. (FBI 2) sp. (FD17) sp. S2 Enzymehydrolyzing activity{circumflex over ( )} Amylase − − − − − −Cellulase + − + + ++ + Chitinase − − − + + − Hemolytic + + − + + n.d.Lipase ++ + + +++ ++ − Pectinase − + − + + − Phosphatase − ++ − ++ +++ −Protease − − − − − + Xylanase + − +++ + ++ + Bacillus PantoeaAcinetobacter Paenibacillus Characteristics sp. S4 sp. S6 sp. S9 sp. S10Enzyme hydrolyzing activity{circumflex over ( )} Amylase − − − +Cellulase + − − + Chitinase + − − − Hemolytic n.d. n.d. n.d. n.d.Lipase + + + + Pectinase − + − + Phosphatase − + − + Protease + − − −Xylanase + + − +

All strains showed lipase activity, while only S10 produced amylaseactivity. S2 and S4 produced significant protease activity. Pectinaseand phosphatase activity was observed with strains FF34, FB12, FD17, S6and S10. All strains were positive for cellulase and/or xylanase exceptstrains FF34 and S9. Chitinase was produced by FB12, FD17 and S4strains, while all strains tested except for FC42 showed hemolyticactivity.

Antagonistic Activities Against Plant Pathogenic Bacteria, Fungi andOomycetes

The antagonistic activities of bacterial isolates were screened againstplant pathogenic bacteria (Agrobacterium tumefaciens, Pseudomonassyringae, Streptococcus pneumoniae), fungi (Fusarium caulimons, Fusariumgraminarium, Fusarium oxysporum, Fusarium solani, Rhizoctonia solani,Thielaviopsis basicold) and oomycetes (Phytophthora infestans,Phytophthora citricola, Phytophthora cominarum). For antibacterialassays, the bacterial isolates and pathogen were cultivated in trypticsoy broth at 30° C. for 24 h. The bacterial isolates werespot-inoculated (10 μL aliquots) on TSA plates pre-seeded with 100 μLtested pathogen. The plates were incubated at 28° C. for 48 h and clearzones of inhibition were recorded.

Antagonistic activity of the bacterial isolates against fungi andoomycetes was tasted by the dual culture technique on potato dextroseagar (PDA) and yeast malt agar (YMA) media (Dennis and Webster 1971,Trans Brit Mycol Soc 57:25-39, incorporated herein by reference). Asmall disk (5 mm) of target fungus/oomycetes was placed in the center ofpetri dishes of both media. Aliquots of 10 μL of overnight bacterialcultures grown in tryptic soy broth were spotted 2 cm away from thecenter. Plates were incubated for 14 days at 24° C. and zones ofinhibition were scored.

TABLE 6 Antimicrobial Activity by Endophytic Bacteria AgrobacteriumPantoea Sphinogobium Pseudomonas Enterobacter Micrococus Characteristicssp. (FA13) sp. (FF34) sp. (FC42) sp. (FBI 2) sp. (FD17) sp. S2Anti-bacterial activity A. tumefaciens − − − ++ + − E. coli n.d. n.d.n.d. n.d. n.d. + P. syringae − − − +++ + − Saureus − − − + − +Anti-fungal activity F. caulimons ++ + + ++ +++ − F. grammarium + + + +++ F. oxysporum + ++ + ++ ++ + F. solani ++ + ++ ++ +++ − R.solani + + + ++ ++ + T. basicola + + + ++ + − Anti-oomycete activity P.infestans + + + ++ ++ − P. citricola + + + ++ +++ − P. cominarum + + +++ ++ Bacillus Pantoea Acinetobacter Paenibacillus Characteristics sp.S4 sp. S6 sp. S9 sp. S10 Anti-bacterial activity A. tumefaciens + − − +E. coli + − − + P. syringae + − − + Saureus + + + + Anti-fungal activityF. caulimons + + − + F. grammarium + + + F. oxysporum + + F. solani + −− + R. solani + + + + T. basicola + + − + Anti-oomycete activity P.infestans − + − − P. citricola − + + + P. cominarum + + + +Effect of Endophytic Strains on Maize Germination

Inoculants of the selected strains were prepared in 50 mL tryptic soybroth in 100 mL Erlenmeyer flasks and incubated at 28±2° C. for 48 h inthe orbital shaking incubator (VWR International, GmbH) at 180 r min⁻¹.The optical density of the broth was adjusted to 0.5 measured at 600 nmusing spectrophotometer (Gene Quant Pro, Gemini BV, The Netherlands) toobtain a uniform population of bacteria (10⁸-10⁹ colony-forming units(CFU) mL⁻¹) in the broth at the time of inoculation. Morescientifically, harvested bacterial cells could be resuspended in thephosphate buffered saline. The inoculum density adjusts using aspectrophotometer to achieve population density (Pillay and Nowak 1997,Can J Microbiol 43:354-361, incorporated herein by reference).

Maize seeds were surface-sterilized with 70% ethanol (3 min), treatedwith 5% NaOHCl for 5 min, and followed by washing 3 times with steriledistilled water (1 min each time). The efficacy of surface sterilizationwas checked by plating seed, and aliquots of the final rinse onto LBplates. Samples were considered to be successfully sterilized when nocolonies were observed on the LB plates after inoculation for 3 days at28° C. Surface-disinfected seeds of different maize cultivars (Helmi,Morignon, Pelicon, Peso and Cesor) were immersed in the bacterialsuspensions for 30 min. The bacterized seeds were deposited onto softwater-agar plates (0.8%, w/v agar) and plates were placed in the dark atroom temperature (24±2° C.). After 96 hrs the percentage of germinatedseeds was scored. Surface-sterilized seeds, but not bacterized (treatedin tryptic soy broth), served as the germination control.

Inoculation of maize seeds with endophytic bacteria increased thegermination rate of all cultivars by 20-40% compared to theun-inoculated control. Maximum increase was observed by inoculation withstrain FD17 (40%) in maize cv. Morignon followed by strains FF34, FA13,FB12 and FC42 (data not shown).

In other experiments, seeds of different cultivars of Maize (Palazzo &die Samba), and Tomato (Red Pear and Gartenfreund) were used to test forpromotion of germination. The results, provided below in Table 7, showthat virtually all strains show a marked increase in germination rates.For maize, Palazzo seeds inoculated with the strains FA13, FF34, S2, S6,S9 and S10 show greater than 90% germination after four days, as did dieSamba seeds inoculated with FF34 and S9 seeds. For tomato, Red Pearseeds inoculated with the strains FB12, FF34, S6 and S10 showed 90% orgreater germination rate after 12 days.

TABLE 7 Germination rate of maize and tomato seeds inoculated withendophytes Maize Germination Rate Tomato Germination Rate (4 Days) (12days) Maize Maize Tomato Tomato Strain “Palazzo” “die Samba” “Red Pear”“Gartenfreund” Neg. control 73.3% 73.3% 33.3% 50.0% FA13 100.0% 86.7%83.3% 60.0% FB12 83.3% 76.7% 96.7% 53.3% FC42 86.7% 86.7% 76.7% 80.0%FD17 76.7% 66.7% 43.3% 46.7% FF34 93.3% 93.3% 96.7% 50.0% S2 93.3% 70.0%70.0% 60.0% S4 70.0% 86.7% 76.7% 66.7% S6 90.0% 80.0% 100.0% 70.0% S996.7% 96.7% 60.0% 53.3% S10 93.3% 80.0% 90.0% 76.7%In Vitro Screening of Efficient Strains Under Axenic Conditions

A growth chamber experiment was conducted on maize to screen theselected strains for their growth promoting activity under gnotobioticconditions. We used specially designed glass tubes with beaded rim(Duran group, DURAN GmbH, Mainz, Germany) for the experiment. The glasstubes were covered with lid to generate fully axenic conditions (noexposure to any environmental factors). Bacterial inoculant productionand seed treatment were done as described above. As control, seeds weretreated with sterilized tryptic soy broth. Treated seeds were placedonto water-agar plates for germination. After 5 days, germinatedseedlings (3-5 cm long) were transferred in the sterilized glass tubescontaining sterilized 20 ml MS (Murashige and Skoog) medium (DuchefaBiochemie, The Netherlands) (4.8 g L⁻¹) and placed at 25±2° C. set at a16 h light and 8 h dark period, with a light intensity of 350 μmol m⁻²s⁻¹. Data regarding shoot/root length and biomass were recorded after 24days. Colonization of inoculant strains was scored by re-isolation ofendophytes. One g of plant shoot was homogenized with a pestle andmortar in 4 ml of 0.9% (w/v) NaCl solution. The number of cultivableendophytes in maize shoot, expressed in CFU per gram (fresh weight), wasdetermined by spreading serial dilution up to 10⁻⁴ (0.1 mL) ofhomogenized surface-sterilized plant material onto TSA (DIFCOLaboratories, Detroit, Mich.) agar medium. Four replicates for eachtreatment were spread on the agar plates and incubated for 5 days at 28°C. Twenty colonies per treatment were randomly selected and theiridentity with the inoculant strain was confirmed by restriction fragmentlength polymorphism (RFLP) analysis of the 16S-23S rRNA intergenicspacer (IGS) region (Reiter et al. 2001, Appl Environ Microbiol68:2261-2268, incorporated herein by reference).

All strains significantly increased the seedling growth compared to thecontrol. As shown in FIGS. 1A-1C, all strains significantly promotedbiomass production, with increases in both root, shoot or overallbiomass. Though responses were variable, the strains generally increasedroot and shoot length in all three cultivars of maize tested.

Next, colonization of plants was tested for all bacterial strains. Asshown in Table 8, strains FA13, FF34, FC42, FB12 and FD17 successfullycolonized corn plants, showing successful colonization of the variousstrains, as detected in the shoot tissue of various cultivars of maize.The amount of detectable bacteria in the shoot tissue varied, rangingfrom 1.58×10⁴ in FB12-inoculated Helmi cultivar, to 1.83×10⁷ CFU foundin Peso cultivars inoculated with FF34. Therefore, the microbesdescribed herein, when contacted with seeds of plants, are capable ofcolonizing the plant as detectable, in this case, in the shoot tissue.Furthermore, colonization of Kolea, Mazurka and DaSilvie cultivars ofmaize by strains S2, S4, S6, S9 and S10 was confirmed by isolatingbacterial cells from homogenates of surface sterilized shoot tissue ofplants grown from inoculated seeds on tryptic soy agar plates for twodays on 28° C. and testing the identity of colonies with IGS regionsequencing to confirm the presence of the microbe. S2, S4, S6, S9 andS10 strains were successfully recovered from the tissues of thesecultivars (data not shown).

TABLE 8 Colonization of Maize Plants by Endophytic Bacteria StrainsHelmi Peso Pelicon Morignon Cesor FA13 1.95 × 10³ 1.16 × 10⁷  1.2 × 10⁴1.21 × 10⁶ 3.31 × 10⁶ FF34 2.66 × 10⁶ 1.83 × 10⁷ 1.21 × 10⁵ 4.13 × 10⁶ 9.1 × 10⁶ FC42 4.63 × 10⁵ 1.37 × 10⁶ 2.00 × 10⁴ 8.24 × 10⁶ 1.07 × 10⁵FB12 1.58 × 10⁴ 1.94 × 10⁷ 1.12 × 10³ 1.46 × 10⁶ 9.38 × 10³ FD17 1.92 ×10⁶ 2.60 × 10⁷ 1.44 × 10⁷ 2.93 × 10⁷ 1.73 × 10⁶Stomatal Conductance and Photosynthesis Rates

Maize plants inoculated with the strains described herein were testedfor photosynthesis and stomatal conductance. As shown in FIG. 2, maizeplants inoculated with the strains display an increase in stomatalconductance when compared with uninoculated controls (ranging from a 36%to 49%) increase), with S2, S6, S9 strains displaying the highest levelof conductance. Therefore, there is an appreciable increase in stomatalconductance conferred by the bacterial of the present invention.

Strain-inoculated maize plants were also tested for photosyntheticrates. As shown in FIG. 3, all strains conferred increasedphotosynthesis rates when compared with control plants in all threemaize cultivars tested (DaSilvie, Mazurka, and Kolea cultivars; averageof three cultivars shown), with an increase ranging from 17% overcontrols (for S9 and S10 strains) to over 23% over controls (S6 strain).Therefore, the endophytic bacterial strains described above conferincreased photosynthesis rates on the host plants.

Net-House Experiment

On the basis of the results from tests performed under axenicconditions, strain FD17 was selected for further evaluation in a pottrial, in which plants were grown in large containers exposed to naturalenvironmental conditions.

Maize plants were grown in soil collected from agricultural (maize)fields in Fischamend, Lower Austria, Austria. The soil was silty clayloam and had the following characteristics: 12% sand, 61% silt, 27%)clay, pH 6.5, 3.3% total carbon, 0.18% total nitrogen, 0.13 mg g⁻¹available phosphorus, 0.066 mg g⁻¹ extractable potassium.

Surface-disinfected seeds of two maize cultivars (Morignon and Peso)were immersed in bacterial suspension (prepared as described above) for1 h. For the un-inoculated control, seeds were treated with sterilizedtryptic soy broth. Seeds were sown in a plastic tray (wiped withethanol) and 12 days old seedlings were transferred into containersfilled with 45 kg soil (2 plants in each container) and placed in anet-house and exposed to natural environmental conditions.

Weather conditions i.e. precipitation, temperature and relative humiditywere recorded by ‘Zentralanstalt fur Meteorologie and Geodynamik’ (ZAMG)during the crop growth period and described in FIGS. 1A-1C. There werethree replicates and the pots were arranged in a completely randomizeddesign. Recommended dose of NPK fertilizers (160-100-60 kg ha⁻¹) wereapplied in each container and tap water was applied to the container forirrigation whenever needed. Data of photochemical efficiency of PSII wasrecorded at flowering stage using handy PEA (Hansatech Instruments Ltd.England) in the mid of July where day time temperature varied from30-35° C. The PSII efficiency in terms of F_(v)/F_(m) was calculatedfrom the data. Growth and yield contributing parameters were recorded atmaturity. The plants were harvested 140 days after planting. FIG. 4shows the PS II efficiency of maize plants inoculated with the bacterialendophyte populations described herein.

Maize plants inoculated with the bacterial endophytes S2, S4, S6, S9,S10 and FD17 were tested for increased leaf area. As shown in FIG. 5,and in Table 9, all the tested strains increased the leaf areasignificantly over the controls.

Similarly, maize plants inoculated with the strains showed a dramaticincrease in chlorophyll content (FIG. 6) over control plants, with thehighest levels found in S6 inoculated plants.

Table 9 below shows the effect of FD17 inoculation on the physiology,growth parameters and yield of two maize cultivars grown in field soiland exposed to natural climatic conditions. Inoculation with strain FD17led to a significant increase in leaf area of both cultivars (20% and13%), respectively). Similarly, biomass (leaf dry weight) was increasedby 27% and 23% in the cultivars Peso and Morignon, respectively, ascompared to the control. In addition, root and plant dry biomass andplant height were significantly enhanced, as was the average cob weight(35% and 42% increase in Peso and Morignon, respectively, as compared tocontrol). The FD17 strain also significantly affected other plantphysiological characteristics: for example, there was a significantincrease in chlorophyll fluorescence (up to a 9% in the Peso cultivar)and a shortened time before onset of flowering (up to 10 days incultivar Peso).

TABLE 9 Effect of inoculation with endophytic strain FD17 on physiology,growth parameters and yield of two maize cultivars grown in pots infield soil and exposed to natural climatic conditions (net houseexperiment) Treatment Peso Morignon Un- Inoculated Un- InoculatedParameters inoculated with FD17 inoculated with FD17 Fv/Fm 0.69 0.750.73 0.79 Time to onset of 65.33 55 70.67 66.33 flowering (days) Plantheight (cm) 192.33 208 196.69 213.68 No. of leaves plant 12.33 14 13.1714.67 Leaf area (cm) 494.26 556.27 512.39 617.11 Leaf dry weight (g)22.21 28.16 28.09 34.56 Plant dry biomass (g) 114.18 153.77 160.46223.14 Root dry biomass (g) 17.26 24.34 19.73 28.28 Cob weight (g)115.28 155.83 123.71 176.23

Rhizosphere and endophytic colonization of roots, stems and leaves bythe inoculant strain were determined by plate counting using TSA plates.Root, stem and leave samples were washed, surface sterilized (asdescribed above) and used for inoculant strain recovery (colonization).For this, samples were crushed in 0.9% (w/v) NaCl solution, shaked witha pulsifier (Microgen Bioproducts Ltd., UK) for 30 sec and differentdilutions were spread on TSA plates. Bacterial colonies were countedafter 4 days of incubation at 28±2° C. The selected colonies wereidentified and confirmed by IGS region-based RFLP analysis.

The ability of strain FD17 to colonize various tissues of the hostplant, as well as the rhizosphere surrounding the plant, was examined.As shown in Table 10 below, seeds of two different maize cultivarsinoculated with the FD17 strain resulted in effective, detectablecolonization in the root, shoot and leaf interior. Therefore, the seedswere treated with an amount of the endophytic bacterium that issufficient to colonize the leaf, root, and shoot tissues. Surprisingly,the rhizosphere also had significant levels of detectable FD17. Thissuggests that the beneficial effects of endophytic bacterial strainssuch as FD17 could be exerting effects externally to the plant.

As described elsewhere, the bacteria described herein are capable ofproducing compounds which allow increased availability of limitingnutrients such as phosphate and iron. The strains could be present onthe surface of the seeds in an amount sufficient to efficiently colonizethe plant, but also the surrounding rhizosphere. The presence ofsignificant amounts of detectable bacteria in the rhizosphere raises theinteresting possibility that the seeds can be treated with the microbeseither on its surface or inside the seed in an amount sufficient toalter the rhizosphere of the plant, thereby altering the soil around theplant, and rendering it more hospitable for the plant.

TABLE 10 Colonization of strain FD17 in rhizosphere, root, stem andleaves of two maize cultivars (wire-house experiment) Plant compartmentRoot Shoot Leaf Maize Rhizosphere (cfu interior (cfu interior (cfuinterior (cfu cv. g⁻¹ dry wt) g⁻¹ dry wt) g⁻¹ dry wt) g⁻¹ dry wt) Peso4.07 × 10⁴ 3.39 × 10⁴ 1.63 × 10³ 1.16 × 10² Morignon 9.85 × 10⁴ 8.59 ×10⁴ 3.72 × 10³ 6.23 × 10²Statistical Analyses

The data of plant growth parameters and colonization were subjected toanalyses of variance. The means were compared by Least SignificantDifference (LSD) test (p<0.05) to detect statistical significance amongtreatment (Steel et al. 1997, Principles and procedures of statistics: Abiometrical approach. 3rd ed. McGraw-Hill Book Int. Co., Singapore,incorporated herein by reference). All of the statistical analyses wereconducted using SPSS software version 19 (IBM SPSS Statistics 19, USA).

The invention claimed is:
 1. A method for preparing a corn seedcomprising an endophytic bacterial population, said method comprisingapplying to an exterior surface of the corn seed a formulationcomprising an endophytic bacterial population consisting essentially ofa Paenibacillus species of bacterium comprising a 16S rRNA comprising anucleic acid sequence consisting of SEQ ID NO:10, wherein the endophyticbacterial population is applied in an amount effective to colonize shoottissue of a plant grown from the corn seed comprising the endophyticbacterial population.
 2. The method of claim 1, wherein the formulationfurther comprises at least one member selected from the group consistingof an agriculturally compatible carrier, a tackifier, a microbialstabilizer, a fungicide, an antibacterial agent, an herbicide, anematicide, an insecticide, a plant growth regulator, a rodenticide, anda nutrient.
 3. The method of claim 1, the formulation comprising10{circumflex over ( )}8-10{circumflex over ( )}9 colony forming units(CFU) of the endophytic bacterial population per mL.
 4. A method forconferring one or more fitness benefits to an agricultural corn plantcomprising: a. Providing a corn seed of the corn plant; b. Contactingthe exterior surface of the seed with a formulation comprising anexogenous endophytic bacterial population consisting essentially of aPaenibacillus species of bacterium comprising a 16S rRNA comprising anucleic acid sequence consisting of SEQ ID NO:10, wherein the exogenousendophytic bacterial population is disposed on an exterior surface ofthe seed or seedling in an amount effective to colonize the matureplant, wherein the formulation further comprises at least one memberselected from the group consisting of an agriculturally compatiblecarrier, a tackifier, a microbial stabilizer, a fungicide, anantibacterial agent, an herbicide, a nematicide, an insecticide, a plantgrowth regulator, a rodenticide, and a nutrient; and c. Allowing theseed to grow to a plant under conditions that permit the endophyticbacterium to colonize the plant.
 5. The method of claim 4, wherein theone or more of the fitness benefits are selected from the groupconsisting of increased germination, increased biomass, increasedflowering time, increased plant biomass, increased fruit or grain yield,increased biomass of the fruit or cob, and increased drought tolerancecompared to a plant grown from a non-contacted seed under the sameconditions.
 6. The method of claim 4, the formulation comprising10{circumflex over ( )}8-10{circumflex over ( )}9 colony forming units(CFU) of the endophytic bacterial population per mL.
 7. The method ofclaim 4, wherein the fitness benefit is increased photosynthetic ratesby at least 17%.
 8. The method of claim 4, wherein the fitness benefitis increased rate of seed germination by at least 20%.
 9. The method ofclaim 4, wherein the fitness benefit is increased root biomass.
 10. Themethod of claim 4, wherein the fitness benefit is increased totalbiomass.
 11. The method of claim 4, wherein the fitness benefit isincreased stomatal conductance.
 12. The method of claim 4, wherein thefitness benefit is increased photochemical efficiency.
 13. The method ofclaim 4, wherein the fitness benefit is increased leaf area.
 14. Themethod of claim 4, wherein the fitness benefit is increased chlorophyllcontent.